ABA-responsive Element Research Articles

Overexpression of Medicago sativa LEA4-4 can improve the salt, drought, and oxidation resistance of transgenicArabidopsis.

Late embryogenesis abundant (LEA) proteins are widely involved in many adverse conditions among plants. In this study, we isolated a LEA4 gene from alfalfa (Medicago sativa L.) termed MsLEA4-4 via a homology cloning strategy. MsLEA4-4 encodes 166 amino acids, and the structural analysis showed that the gene contained five repeating TAQAAKEKTQQ amino acid motifs. There were a large number of α-helix in MsLEA4-4, and belongs to hydrophilic amino acid. Subcellular localization analysis showed that MsLEA4-4 was localized in the nucleus. The MsLEA4-4 promoter consisted of G-box and A-box elements, abscisic acid-responsive elements (ABREs), photo regulation and photoperiodic-controlling cis-acting elements, and endosperm expression motifs. The MsLEA4-4 overexpressing in Arabidopsis conferred late-germination phenotypes. Resistance of the overexpressed plants to abiotic stress significantly outperformed the wild-type (WT) plants. Under salt stress and abscisic acid treatment, with more lateral roots and higher chlorophyll content, the overexpressed plants has a higher survival rate measured against WT. Compared to those in the WT plants, the levels of soluble sugar and the activity of various antioxidant enzymes were elevated in the overexpressed plants, whereas the levels of proline and malondialdehyde were significantly reduced. The expression levels of several genes such as ABF3, ABI5, NCED5, and NCED9 increased markedly in the overexpressed plants compared to the WT under osmotic stress.

Transcriptomic analysis of poco1, a mitochondrial pentatricopeptide repeat protein mutant in Arabidopsis thaliana

BackgroundFlowering is a crucial stage during plant development. Plants may respond to unfavorable conditions by accelerating reproductive processes like flowering. In a recent study, we showed that PRECOCIOUS1 (POCO1) is a mitochondrial pentatricopeptide repeat (PPR) protein involved in flowering time and abscisic acid (ABA) signaling in Arabidopsis thaliana. Here, we use RNA-seq data to investigate global gene expression alteration in the poco1 mutant.ResultsRNA-seq analysis was performed during different developmental stages for wild-type and poco1 plants. The most profound differences in gene expression were found when wild-type and poco1 plants of the same developmental stage were compared. Coverage analysis confirmed the T-DNA insertion in POCO1, which was concomitant with truncated transcripts. Many biological processes were found to be enriched. Several flowering-related genes such as FLOWERING LOCUS T (FT), which may be involved in the early-flowering phenotype of poco1, were differentially regulated. Numerous ABA-associated genes, including the core components of ABA signaling such as ABA receptors, protein phosphatases, protein kinases, and ABA-responsive element (ABRE) binding proteins (AREBs)/ABRE-binding factors (ABFs) as well as important genes for stomatal function, were mostly down-regulated in poco1. Drought and oxidative stress-related genes, including ABA-induced stress genes, were differentially regulated. RNA-seq analysis also uncovered differentially regulated genes encoding various classes of transcription factors and genes involved in cellular signaling. Furthermore, the expression of stress-associated nuclear genes encoding mitochondrial proteins (NGEMPs) was found to be altered in poco1. Redox-related genes were affected, suggesting that the redox state in poco1 might be altered.ConclusionThe identification of various enriched biological processes indicates that complex regulatory mechanisms underlie poco1 development. Differentially regulated genes associated with flowering may contribute to the early-flowering phenotype of poco1. Our data suggest the involvement of POCO1 in the early ABA signaling process. The down-regulation of many ABA-related genes suggests an association of poco1 mutation with the ABA signaling deficiency. This condition further affects the expression of many stress-related, especially drought-associated genes in poco1, consistent with the drought sensitivity of poco1. poco1 mutation also affects the expression of genes associated with the cellular regulation, redox, and mitochondrial perturbation.

Wheat TabZIP8, 9, 13 participate in ABA biosynthesis in NaCl-stressed roots regulated by TaCDPK9-1

In plants, basic leucine zipper (bZIP) transcription factors (TFs) participate in various biological processes such as development and stress responses. But the molecular mechanism of wheat bZIP TFs modulating abscisic acid (ABA) biosynthesis is unknown. In this study, we demonstrated the expressions of three bZIP TF genes TabZIP8, 9, 13, were regulated by Triticum aestivum calcium (Ca2+)-dependent protein kinase 9–1 (TaCDPK9-1) and they took part in ABA biosynthesis in wheat roots under salt stress. We first isolated TabZIP8, 9, 13 and TaCDPK9-1 from wheat. TabZIP8, 9, 13 genes transcripts were strongly induced by salt stress, but salt-induced TabZIP8, 9, 13 transcriptions were drastically impaired by Ca2+ channel blocker LaCl3. TaCDPK9-1 kinase could interact with TabZIP8, 9, 13 TFs through yeast two-hybrid assay. Next, the expression levels of salt-induced wheat 9-cis-epoxycarotenoid dioxygenase1, 2 (TaNCED1,2) encoding a key enzyme controlling ABA production and salt-induced ABA content were found to be decreased under LaCl3 treatment, and yeast one-hybrid experiment revealed TabZIP8, 9, 13 could bind to the ABA-responsive elements (ABREs) and the promoter sequence of TaNCED2 gene. Together, our results suggest that salt stress-induced ABA accumulation is mediated by TabZIP8, 9, 13, which are adjusted by TaCDPK9-1 in wheat roots.

Trehalose-6-phosphate phosphatase E modulates ABA-controlled root growth and stomatal movement in Arabidopsis.

Trehalose plays important roles in plant growth and stress responses and is synthesized from trehalose‐6‐phosphate by trehalose‐6‐phosphate phosphatase (TPP). Here, we show that trehalose and abscisic acid (ABA) have synergistic effects on root growth and stomatal closure. The Arabidopsis thaliana genome contains ten genes encoding TPPs and the expression level of one, TPPE, and trehalose contents increased in response to ABA. In the presence of ABA, the ABA‐responsive transcription factor ABA RESPONSE ELEMENT BINDING FACTOR2 (ABF2) directly binds to the TPPE promoter to activate its expression. Genetic analysis revealed that TPPE acts downstream of ABF2, which is supported by the findings that TPPE expression and trehalose content are reduced in the abf2 mutant and that a mutation in TPPE abolished the ABA‐sensitive root elongation phenotype of 35S:ABF2 plants. Reactive oxygen species (ROS) accumulation in response to ABA failed to occur in tppe mutant plants, suggesting that TPPE is involved in ABA‐controlled root elongation and stomatal movement by inducing ROS accumulation. This study uncovers a new branch of the ABA signaling pathway and provides a molecular basis for the role of trehalose in plant responses to abiotic stress.

OsMFT2 is involved in the regulation of ABA signaling-mediated seed germination through interacting with OsbZIP23/66/72 in rice.

Seed germination is a complex process involving various physical and biochemical cues, determined by exogenous and endogenous factors. Here, we identified a gene, OsMFT2, that negatively regulates seed germination in rice. OsMFT2 knock-out lines exhibited pre-harvest sprouting, whereas OsMFT2 overexpression lines showed delayed germination. RNA expression profiling showed that OsMFT2 was specifically expressed in seeds. Subcellular localization indicated that OsMFT2 was a nuclear protein. Exogenous abscisic acid (ABA) treatment of imbibed seeds and seedlings indicated that OsMFT2 altered ABA sensitivity during seed germination and post-germination growth. In vivo and in vitro assays showed that three bZIP transcription factors, OsbZIP23, OsbZIP66 and OsbZIP72, interacted with OsMFT2. OsbZIP23/66/72 bound to the promoter of Rab16A, a typical gene containing the ABA-responsive element, and OsMFT2 enhanced the binding to the Rab16A promoter. Moreover, several ABA-responsive genes were differentially expressed in the imbibed seeds of OsMFT2 transgenic lines and the wild type. The performance of the transgenic plants demonstrated that overexpressing OsbZIP23 rescued the pre-harvest sprouting phenotype and the decrease in ABA-signaling genes expression caused by OsMFT2 knock-out. All of these results demonstrate that OsMFT2 positively regulates ABA-responsive genes through interacting with OsbZIP23/66/72 and functions in seed germination.

Exogenous Abscisic Acid Supplementation at Early Stationary Growth Phase Triggers Changes in the Regulation of Fatty Acid Biosynthesis in Chlorella vulgaris UMT-M1.

Abscisic acid (ABA) has been known to exist in a microalgal system and serves as one of the chemical stimuli in various biological pathways. Nonetheless, the involvement of ABA in fatty acid biosynthesis, particularly at the transcription level in microalgae is poorly understood. The objective of this study was to determine the effects of exogenous ABA on growth, total oil content, fatty acid composition, and the expression level of beta ketoacyl-ACP synthase I (KAS I) and omega-3 fatty acid desaturase (ω-3 FAD) genes in Chlorella vulgaris UMT-M1. ABA was applied to early stationary C. vulgaris cultures at concentrations of 0, 10, 20, and 80μM for 48h. The results showed that ABA significantly increased biomass production and total oil content. The increment of palmitic (C16:0) and stearic (C18:0) acids was coupled by decrement in linoleic (C18:2) and α-linolenic (C18:3n3) acids. Both KAS I and ω-3 FAD gene expression were downregulated, which was negatively correlated to saturated fatty acid (SFAs), but positively correlated to polyunsaturated fatty acid (PUFA) accumulations. Further analysis of both KAS I and ω-3 FAD promoters revealed the presence of multiple ABA-responsive elements (ABREs) in addition to other phytohormone-responsive elements. However, the role of these phytohormone-responsive elements in regulating KAS I and ω-3 FAD gene expression still remains elusive. This revelation might suggest that phytohormone-responsive gene regulation in C. vulgaris and microalgae as a whole might diverge from higher plants which deserve further scientific research to elucidate its functional roles.

Molecular characterization of the promoter of the stress-inducible ZmMYB30 gene in maize

The ZmMYB30 is a member of the myeloblastosis (MYB) transcription factor superfamily, which has been shown to be a transcription regulator in abiotic stress tolerance in maize (Zea mays). To further identify the biological function of ZmMYB30 and reveal how its expression is induced in response to stress, we isolated the ZmMYB30 promoter and conducted a functional analysis. A 1461-bp promoter fragment was cloned and sequenced. Motif prediction using PlantCARE revealed several stress-responsive elements in the promoter sequence. Deletions in the promoter sequence affected the activity of the promoter and demonstrated that putative stress-responsive motifs, including TC-rich repeats (ATTCTCTAAC), abscisic acid responsive element (ABRE, ACGTG), and MYB binding site (MBS, CAACTG), played important roles in regulating the expression of ZmMYB30. Promoter β-glucuronidase (GUS) analysis also showed that GUS expression under the control of the ZmMYB30 promoter responded to drought and salinity. Many natural variations in the ZmMYB30 promoter sequence were found among 31 inbred maize lines, including 22 single nucleotide polymorphisms (SNPs), 17 insertion and deletion (InDels), and approximately 400-bp long deletions. The ZmMYB30 expression varied greatly among the different inbred lines. The long fragment deletion in the promoter region did not impair the ZmMYB30 expression, whereas SNP (-374) in the MBS motif in the 62R and LH82 lines greatly decreased the ZmMYB30 expression. A site-specific mutation in the MBS motif also decreased the expression of the reporter GUS gene driven by the ZmMYB30 promoter sequence. The expressions of three stress-responsive genes ZmSOS1, ZmSOS2, and ZmABF3 were found to be consistent with the ZmMYB30 expression. Our results provide new evidence to support the role of ZmMYB30 as an important regulator in maize stress tolerance.

TaABI5, a wheat homolog of Arabidopsis thaliana ABA insensitive 5, controls seed germination

Abscisic acid (ABA) response element (ABRE)-binding factors (ABFs) are basic region/leucine zipper motif (bZIP) transcription factors that regulate the expression of ABA-induced genes containing ABRE in their promoters. The amino acid sequence of the wheat bZIP protein, TaABI5, showed high homology to that of Arabidopsis ABA insensitive 5 (ABI5). TaABI5 was classified into the clade of ABI5s in Arabidopsis and rice, unlike TRAB1 of rice, Wabi5 of wheat, and HvABI5 of barley in the bZIP Group A family, by a phylogenetic analysis. TaABI5 was strongly expressed in seeds during the late ripening and maturing stages; however, its expression level markedly decreased after germination. An in situ hybridization analysis showed that TaABI5 mRNA accumulated in seed embryos, particularly the scutellum. In a transient assay using wheat aleurone cells, TaABI5 activated the promoter of Em containing ABRE, which is an embryogenesis abundant protein gene, indicating that TaABI5 acts as a transcription factor in wheat seeds. Furthermore, the seeds of transgenic Arabidopsis lines introduced with 35S:TaABI5 exhibited high sensitivity to ABA and the inhibition of germination. The seed dormancy of the transgenic Arabidopsis lines was stronger than that of Col. These results support TaABI5 playing an important role in mature seeds, particularly before seed germination, and acting as a functional ortholog to Arabidopsis ABI5.

GAI Functions in the Plant Response to Dehydration Stress in Arabidopsis thaliana.

DELLA (GAI/RGA/RGL1/RGL2/RGL3) proteins are key negative regulators in GA (gibberellin) signaling and are involved in regulating plant growth as a response to environmental stresses. It has been shown that the DELLA protein PROCERA (PRO) in tomato promotes drought tolerance, but its molecular mechanism remains unknown. Here, we showed that the gai-1 (gibberellin insensitive 1) mutant (generated from the gai-1 (Ler) allele (with a 17 amino acid deletion within the DELLA domain of GAI) by backcrossing gai-1 (Ler) with Col-0 three times), the gain-of-function mutant of GAI (GA INSENSITIVE) in Arabidopsis, increases drought tolerance. The stomatal density of the gai-1 mutant was increased but its stomatal aperture was decreased under abscisic acid (ABA) treatment conditions, suggesting that the drought tolerance of the gai-1 mutant is a complex trait. We further tested the interactions between DELLA proteins and ABF2 (abscisic acid (ABA)-responsive element (ABRE)-binding transcription factors) and found that there was a strong interaction between DELLA proteins and ABF2. Our results provide new insight into DELLA proteins and their role in drought stress tolerance.

Combinatorial interactions of the LEC1 transcription factor specify diverse developmental programs during soybean seed development

The LEAFY COTYLEDON1 (LEC1) transcription factor is a central regulator of seed development, because it controls diverse biological programs during seed development, such as embryo morphogenesis, photosynthesis, and seed maturation. To understand how LEC1 regulates different gene sets during development, we explored the possibility that LEC1 acts in combination with other transcription factors. We identified and compared genes that are directly transcriptionally regulated by ABA-RESPONSIVE ELEMENT BINDING PROTEIN3 (AREB3), BASIC LEUCINE ZIPPER67 (bZIP67), and ABA INSENSITIVE3 (ABI3) with those regulated by LEC1. We showed that LEC1 operates with specific sets of transcription factors to regulate different gene sets and, therefore, distinct developmental processes. Thus, LEC1 controls diverse processes through its combinatorial interactions with other transcription factors. DNA binding sites for the transcription factors are closely clustered in genomic regions upstream of target genes, defining cis-regulatory modules that are enriched for DNA sequence motifs that resemble sequences known to be bound by these transcription factors. Moreover, cis-regulatory modules for genes regulated by distinct transcription factor combinations are enriched for different sets of DNA motifs. Expression assays with embryo cells indicate that the enriched DNA motifs are functional cis elements that regulate transcription. Together, the results suggest that combinatorial interactions between LEC1 and other transcription factors are mediated by cis-regulatory modules containing clustered cis elements and by physical interactions that are documented to occur between the transcription factors.

Cultivar variation in hormone- and sugar-response reveals abscisic acid-responsive sucrose phloem loading at the early regenerative stage is a significant determinant of seed yield in Brassica napus

Seed crop yield is determined by manipulating source-sink interaction. To characterize the metabolic and signaling network encompassing plant hormones and sugar, seed yield in two cultivars of Brassica napus was interpreted as being linked to changes in the physiological parameters involved in carbohydrate metabolism and endogenous hormone level from the bolting to pod filling stage. Overall salicylic acid (SA), jasmonic acid (JA), and gibberellic acid (GA) increased, whereas indole-3-acetic acid (IAA) and abscisic acid (ABA) decreased from the bolting to pod filling stage. In both cultivars Akela and Colosse, leaf senescence progressed as evidenced by the loss of photosynthetic pigment and enhanced expression of senescence-related gene SAG12. Leaf senescence parameters were shown to be closely associated with enhanced hexose/sucrose ratio, but not with sucrose. Sucrose content was largely higher at the bolting stage, then decreased significantly at the pod filling stage in an ABA-dependent manner. Sucrose content was parallel with the expression of ABA-responsive element binding 2 (AREB2) and sucrose non-fermenting kinase 2 (SnRK2). Sucrose phloem loading at the bolting stage was shown to be more influential in determining seed yield. In the higher seed yielding cultivar Colosse, the highest sucrose phloem loading with enhanced expression of SUT4 and SWEET11 was reflected in the highest expression of SnRK2 and AREB2 in an ABA-dependent manner. Thus, these results indicate that sucrose transport from mature leaves at the bolting stage, which is eventually regulated by ABA-responsive sucrose signaling genes SnRK2 and AREB2, is a significant determinant of seed yield of oilseed rape.

Overexpression of VIRE2-INTERACTING PROTEIN2 in Arabidopsis regulates genes involved in Agrobacterium-mediated plant transformation and abiotic stresses

Arabidopsis VIRE2-INTERACTING PROTEIN2 (VIP2) was previously described as a protein with a NOT domain, and Arabidopsis vip2 mutants are recalcitrant to Agrobacterium-mediated root transformation. Here we show that VIP2 is a transcription regulator and the C-terminal NOT2 domain of VIP2 interacts with VirE2. Interestingly, AtVIP2 overexpressor lines in Arabidopsis did not show an improvement in Agrobacterium-mediated stable root transformation, but the transcriptome analysis identified 1,634 differentially expressed genes compared to wild-type. These differentially expressed genes belonged to various functional categories such as membrane proteins, circadian rhythm, signaling, response to stimulus, regulation of plant hypersensitive response, sequence-specific DNA binding transcription factor activity and transcription regulatory region binding. In addition to regulating genes involved in Agrobacterium-mediated plant transformation, AtVIP2 overexpressor line showed differential expression of genes involved in abiotic stresses. The majority of the genes involved in abscisic acid (ABA) response pathway, containing the Abscisic Acid Responsive Element (ABRE) element within their promoters, were down-regulated in AtVIP2 overexpressor lines. Consistent with this observation, AtVIP2 overexpressor lines were more susceptible to ABA and other abiotic stresses. Based on the above findings, we hypothesize that VIP2 not only plays a role in Agrobacterium-mediated plant transformation but also acts as a general transcriptional regulator in plants.

Genome-wide identification of and functional insights into the late embryogenesis abundant (LEA) gene family in bread wheat (Triticum aestivum)

Late embryogenesis abundant (LEA) proteins are involved in the responses and adaptation of plants to various abiotic stresses, including dehydration, salinity, high temperature, and cold. Here, we report the first comprehensive survey of the LEA gene family in “Chinese Spring” wheat (Triticum aestivum). A total of 179 TaLEA genes were identified in T. aestivum and classified into eight groups. All TaLEA genes harbored the LEA conserved motif and had few introns. TaLEA genes belonging to the same group exhibited similar gene structures and chromosomal locations. Our results revealed that most TaLEA genes contained abscisic acid (ABA)-responsive elements (ABREs) and various cis-acting elements associated with the stress response in the promoter region and were induced under ABA and abiotic stress treatments. In addition, 8 genes representing each group were introduced into E. coli and yeast to investigate the protective function of TaLEAs under heat and salt stress. TaLEAs enhanced the tolerance of E. coli and yeast to salt and heat, indicating that these proteins have protective functions in host cells under stress conditions. These results increase our understanding of LEA genes and provide robust candidate genes for future functional investigations aimed at improving the stress tolerance of wheat.

Involvement of abscisic acid-responsive element-binding factors in cassava (Manihot esculenta) dehydration stress response

Cassava (Manihot esculenta) is a major staple food, animal feed and energy crop in the tropics and subtropics. It is one of the most drought-tolerant crops, however, the mechanisms of cassava drought tolerance remain unclear. Abscisic acid (ABA)-responsive element (ABRE)-binding factors (ABFs) are transcription factors that regulate expression of target genes involved in plant tolerance to drought, high salinity, and osmotic stress by binding ABRE cis-elements in the promoter regions of these genes. However, there is little information about ABF genes in cassava. A comprehensive analysis of Manihot esculenta ABFs (MeABFs) described the phylogeny, genome location, cis-acting elements, expression profiles, and regulatory relationship between these factors and Manihot esculenta betaine aldehyde dehydrogenase genes (MeBADHs). Here we conducted genome-wide searches and subsequent molecular cloning to identify seven MeABFs that are distributed unevenly across six chromosomes in cassava. These MeABFs can be clustered into three groups according to their phylogenetic relationships to their Arabidopsis (Arabidopsis thaliana) counterparts. Analysis of the 5′-upstream region of MeABFs revealed putative cis-acting elements related to hormone signaling, stress, light, and circadian clock. MeABF expression profiles displayed clear differences among leaf, stem, root, and tuberous root tissues under non-stress and drought, osmotic, or salt stress conditions. Drought stress in cassava leaves and roots, osmotic stress in tuberous roots, and salt stress in stems induced expression of the highest number of MeABFs showing significantly elevated expression. The glycine betaine (GB) content of cassava leaves also was elevated after drought, osmotic, or salt stress treatments. BADH1 is involved in GB synthesis. We show that MeBADH1 promoter sequences contained ABREs and that MeBADH1 expression correlated with MeABF expression profiles in cassava leaves after the three stress treatments. Taken together, these results suggest that in response to various dehydration stresses, MeABFs in cassava may activate transcriptional expression of MeBADH1 by binding the MeBADH1 promoter that in turn promotes GB biosynthesis and accumulation via an increase in MeBADH1 gene expression levels and MeBADH1 enzymatic activity. These responses protect cells against dehydration stresses by preserving an osmotic balance that enhances cassava tolerance to dehydration stresses.

Overexpression of the Soybean NAC Gene GmNAC109 Increases Lateral Root Formation and Abiotic Stress Tolerance in Transgenic Arabidopsis Plants

NACs are plant-specific transcription factors that have crucial roles in plant development and biotic and/or abiotic stress responses. This study characterized the functions of the soybean NAC gene GmNAC109 using an overexpression construct in Arabidopsis lines. Sequence analysis revealed that GmNAC109 is highly homologous to ATAF1 (Arabidopsis Transcription Activation Factor 1), which regulates biotic and abiotic stress responses. GmNAC109 protein localized to the nucleus and its C-terminal domain exhibited transcriptional activation activity. Salt, dehydration, and cold stresses significantly increased expression of GmNAC109 in soybean. Similarly, Arabidopsis plants overexpressing GmNAC109 were more tolerant to drought and salt stress than wild-type Col-0 plants. Stress response-related genes, such as DREB1A (drought-responsive element-binding 1A), DREB2A, AREB1 (ABSCISIC ACID-RESPONSIVE ELEMENT BINDING PROTEIN 1), AREB2, RD29A (RESPONSIVE TO Desiccation 29A), and COR15A (COLD REGULATED 15A) were upregulated in GmNAC109-overexpressing transgenic Arabidopsis lines. The transgenic lines showed upregulation of the ABA-responsive genes ABI1 (ABA INSENSITIVE 1) and ABI5 and hypersensitivity to ABA. However, GmNAC109 did not increase expression of the ABA-biosynthetic gene NCED3 (NINE-CIS-EPOXYCAROTENOID DIOXYGENASE 3) and endogenous ABA content in the transgenic lines. Overexpression of GmNAC109 significantly increased lateral root formation in transgenic Arabidopsis lines. Expression of AIR3 (AUXIN-INDUCED IN ROOT CULTURES 3) and ARF2 (AUXIN RESPONSE FACTOR 2) was increased and decreased in these transgenic lines, respectively, indicating that GmNAC109 is involved in the auxin signaling pathway and thereby helps to regulate hairy root formation. Our results provide a basis for development of soybean lines with improved tolerance to abiotic stresses via genetic manipulation.

A role of age-dependent DNA methylation reprogramming in regulating the regeneration capacity of Boea hygrometrica leaves.

Plants can regenerate new individuals under appropriate culture conditions. Although the molecular basis of shoot regeneration has steadily been unraveled, the role of age-dependent DNA methylation status in the regulation of explant regeneration remains practically unknown. Here, we established an effective auxin/cytokinin-induced shoot regeneration system for the resurrection plant Boea hygrometrica via direct organogenesis and observed that regeneration was postponed with increasing age of donor plants. Global transcriptome analysis revealed significant upregulation of genes required for hormone signaling and phenylpropanoid biosynthesis and downregulation of photosynthetic genes during regeneration. Transcriptional changes in the positive/negative regulators and cell wall-related proteins involved in plant regeneration, such as ELONGATED HYPOCOTYL5 (HY5), LATERAL ORGAN BOUNDARIES DOMAIN, SHOOT-MERISTEMLESS, and WUSCHEL, were associated with the regeneration process. Comparison of DNA methylation profiling between leaves from young seedlings (YL) and mature plants (ML) revealed increased asymmetrical methylation in ML, which was predominantly distributed in promoter regions of genes, such as HY5 and a member of ABA-responsive element (ABRE) binding protein/ABRE binding factor, as well as genes encoding glycine-rich cell wall structural protein, CENTRORADIALIS-like protein, and beta-glucosidase 40-like essential for shoot meristem and cell wall architecture. Their opposite transcription response in ML explants during regeneration compared with those from YL demonstrated the putative involvement of DNA methylation in regeneration. Moreover, a significant lower expression of DNA glycosylase-lyase required for DNA demethylation in ML was coincident with its postponed regeneration compared with those in YL. Taken together, our results suggest a role of promoter demethylation in B. hygrometrica regeneration.

Regulation of ABI5 expression by ABF3 during salt stress responses in Arabidopsis thaliana

BackgroundBasic region/leucine zippers (bZIPs) are transcription factors (TFs) encoded by a large gene family in plants. ABF3 and ABI5 are Group A bZIP TFs that are known to be important in abscisic acid (ABA) signaling. However, questions of whether ABF3 regulates ABI5 are still present.ResultsIn vitro kinase assay results showed that Thr-128, Ser-134, and Thr-451 of ABF3 are calcium-dependent protein kinase phosphorylation sites. Bimolecular fluorescence complementation (BiFC) analysis results showed a physical interaction between ABF3 and 14-3-3ω. A Thr-451 to Ala point mutation abolished the interaction but did not change the subcellular localization. In addition, the Arabidopsis protoplast transactivation assay using a luciferase reporter exhibited ABI5 activation by either ABF3 alone or by co-expression of ABF3 and 14-3-3ω. Moreover, chromatin immunoprecipitation-qPCR results showed that in Arabidopsis, ABI5 ABA-responsive element binding proteins (ABREs) of the promoter region (between − 1376 and − 455) were enriched by ABF3 binding under normal and 150 mM NaCl salt stress conditions.ConclusionTaken together, our results demonstrated that ABI5 expression is regulated by ABF3, which could contribute to salt stress tolerance in Arabidopsis thaliana.

Genome-Wide Identification of SNAC1-Targeted Genes Involved in Drought Response in Rice

Drought stress can cause huge crop production losses. Drought resistance consists of complex traits, and is regulated by arrays of unclear networks at the molecular level. A stress-responsive NAC transcription factor gene SNAC1 has been reported for its function in the positive regulation of drought resistance in rice, and several downstream SNAC1 targets have been identified. However, a complete regulatory network mediated by SNAC1 in drought response remains unknown. In this study, we performed Chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA-Seq of SNAC1-overexpression transgenic rice (SNAC1-OE) lines and wild-type under normal and moderate drought stress conditions, to identify all SNAC1 target genes at a genome-wide scale by RNA-Seq analyses. We detected 980 differentially expressed genes (DEGs) in the SNAC1-OE lines compared to the wild-type control under drought stress conditions. By ChIP-Seq analyses, we identified 4,339 SNAC1-binding genes under drought stress conditions (SNAC1BGDs). By combining the DEGs and SNAC1BGDs, we identified 93 SNAC1-targeted genes involved in drought responses (SNAC1TGDs). Most SNAC1TGDs are involved in transcriptional regulation, response to water loss, and other processes related to stress responses. Moreover, the major motifs in the SNAC1BGDs promoters include a NAC recognition sequence (NACRS) and an ABA responsive element (ABRE). SNAC1-OE lines are more sensitive to ABA than wild-type. SNAC1 can bind to the OsbZIP23 promoter, an important ABA signaling regulator, and positively regulate the expression of several ABA signaling genes.

Role of ABA Signaling in Regulation of Stem Sugar Metabolism and Transport under Post- Flowering Drought Stress in Sweet Sorghum

Sugar remobilization from vegetative to reproductive tissues is an important process that determines grain yield in crops. Sweet sorghum stems store sugar and introgression of Stay green1 (Stg 1) locus from the grain sorghum genotype B35 into the sweet sorghum genotype S35 was previously shown to bring about a 2-fold higher stem sugar accumulation in the near-isogenic line (NIL) S35SG06040. We hypothesized that remobilization of stem sugar augments grain yield on exposure to drought stress and that the phytohormone abscisic acid (ABA) has a role to play in this process. ABA levels were three times higher in the NIL as compared to those in S35 on drought stress exposure. Remobilization of stem sugars in the NIL was evident by the observed decrease in reducing sugar content in the stem but not in the peduncle in response to prolonged drought stress exposure. Drought-induced expression of some ABA response factors (ABFs) as well as invertase and sucrose transporter genes was seen to be higher in the NIL as compared to S35. An over-representation of ABA-responsive elements (ABREs) and sugar signaling motifs in the differentially expressed genes indicated the involvement of ABA and sugar signaling in regulation of their expression. Two ABF genes located on the Stg1 locus showed single nucleotide polymorphism, which possibly accounted for their differential regulation in S35 and the NIL. The results suggest that ABA signaling plays an important role in post-flowering drought-induced remobilization of sugars to the reproductive sinks.

Identification of a 119-bp Promoter of the Maize Sulfite Oxidase Gene (ZmSO) That Confers High-Level Gene Expression and ABA or Drought Inducibility in Transgenic Plants.

Drought adversely affects crop growth and yields. The cloning and characterization of drought- or abscisic acid (ABA)-inducible promoters is of great significance for their utilization in the genetic improvement of crop resistance. Our previous studies have shown that maize sulfite oxidase (SO) has a sulfite-oxidizing function and is involved in the drought stress response. However, the promoter of the maize SO gene has not yet been characterized. In this study, the promoter (ZmSOPro, 1194 bp upstream region of the translation initiation site) was isolated from the maize genome. The in-silico analysis of the ZmSOPro promoter identified several cis-elements responsive to the phytohormone ABA and drought stress such as ABA-responsive element (ABRE) and MYB binding site (MBS), besides a number of core cis-acting elements, such as TATA-box and CAAT-box. A 5′ RACE (rapid amplification of cDNA ends) assay identified an adenine residue as the transcription start site of the ZmSO. The ZmSOPro activity was detected by β-glucuronidase (GUS) staining at nearly all developmental stages and in most plant organs, except for the roots in transgenic Arabidopsis. Moreover, its activity was significantly induced by ABA and drought stress. The 5′-deletion mutant analysis of the ZmSOPro in tobacco plants revealed that a 119-bp fragment in the ZmSOPro (upstream of the transcription start site) is a minimal region, which is required for its high-level expression. Moreover, the minimal ZmSOPro was significantly activated by ABA or drought stress in transgenic plants. Further mutant analysis indicated that the MBS element in the minimal ZmSOPro region (119 bp upstream of the transcription start site) is responsible for ABA and drought-stress induced expression. These results improve our understanding of the transcriptional regulation mechanism of the ZmSO gene, and the characterized 119-bp promoter fragment could be an ideal candidate for drought-tolerant gene engineering in both monocot and dicot crops.