Towards the Identification of New Genes Involved in ABA-Dependent Abiotic Stresses Using Arabidopsis Suppressor Mutants of abh1 Hypersensitivity to ABA during Seed Germination

Abscisic acid plays a pivotal role in the abiotic stress response in plants. Although great progress has been achieved explaining the complexity of the stress and ABA signaling cascade, there are still many questions to answer. Mutants are a valuable tool in the identification of new genes or new alleles of already known genes and in elucidating their role in signaling pathways. We applied a suppressor mutation approach in order to find new components of ABA and abiotic stress signaling in Arabidopsis. Using the abh1 (ABA hypersensitive 1) insertional mutant as a parental line for EMS mutagenesis, we selected several mutants with suppressed hypersensitivity to ABA during seed germination. Here, we present the response to ABA and a wide range of abiotic stresses during the seed germination and young seedling development of two suppressor mutants—soa2 (suppressor of abh1 hypersensitivity to ABA 2) and soa3 (suppressor of abh1 hypersensitivity to ABA 3). Generally, both mutants displayed a suppression of the hypersensitivity of abh1 to ABA, NaCl and mannitol during germination. Both mutants showed a higher level of tolerance than Columbia-0 (Col-0—the parental line of abh1) in high concentrations of glucose. Additionally, soa2 exhibited better root growth than Col-0 in the presence of high ABA concentrations. soa2 and soa3 were drought tolerant and both had about 50% fewer stomata per mm² than the wild-type but the same number as their parental line—abh1. Taking into account that suppressor mutants had the same genetic background as their parental line—abh1, it was necessary to backcross abh1 with Landsberg erecta four times for the map-based cloning approach. Mapping populations, derived from the cross of abh1 in the Landsberg erecta background with each suppressor mutant, were created. Map based cloning in order to identify the suppressor genes is in progress.     

Identification of Novel miRNAs and Their Target Genes in the Response to Abscisic Acid in Arabidopsis

miRNAs are involved in various biological processes, including adaptive responses to abiotic stress. To understand the role of miRNAs in the response to ABA, ABA-responsive miRNAs were identified by small RNA sequencing in wild-type Arabidopsis, as well as in abi1td, mkkk17, and mkkk18 mutants. We identified 10 novel miRNAs in WT after ABA treatment, while in abi1td, mkkk17, and mkkk18 mutants, three, seven, and nine known miRNAs, respectively, were differentially expressed after ABA treatment. One novel miRNA (miRn-8) was differentially expressed in the mkkk17 mutant. Potential target genes of the miRNA panel were identified using psRNATarget. Sequencing results were validated by quantitative RT-PCR of several known and novel miRNAs in all genotypes. Of the predicted targets of novel miRNAs, seven target genes of six novel miRNAs were further validated by 5′ RLM-RACE. Gene ontology analyses showed the potential target genes of ABA-responsive known and novel miRNAs to be involved in diverse cellular processes in plants, including development and stomatal movement. These outcomes suggest that a number of the identified miRNAs have crucial roles in plant responses to environmental stress, as well as in plant development, and might have common regulatory roles in the core ABA signaling pathway.     

Genome-wide Identification and Characterization of FCS-Like Zinc Finger (FLZ) Family Genes in Maize (Zea mays) and Functional Analysis of ZmFLZ25 in Plant Abscisic Acid Response

FCS-like zinc finger family proteins (FLZs), a class of plant-specific scaffold of SnRK1 complex, are involved in the regulation of various aspects of plant growth and stress responses. Most information of FLZ family genes was obtained from the studies in Arabidopsis thaliana, whereas little is known about the potential functions of FLZs in crop plants. In this study, 37 maize FLZ (ZmFLZ) genes were identified to be asymmetrically distributed on 10 chromosomes and can be divided into three subfamilies. Protein interaction and subcellular localization assays demonstrated that eight typical ZmFLZs interacted and partially co-localized with ZmKIN10, the catalytic α-subunit of the SnRK1 complex in maize leaf mesophyll cells. Expression profile analysis revealed that several ZmFLZs were differentially expressed across various tissues and actively responded to diverse abiotic stresses. In addition, ectopic overexpression of ZmFLZ25 in Arabidopsis conferred hypersensitivity to exogenous abscisic acid (ABA) and triggered higher expression of ABA-induced genes, pointing to the positive regulatory role of ZmFLZ25 in plant ABA signaling, a scenario further evidenced by the interactions between ZmFLZ25 and ABA receptors. In summary, these data provide the most comprehensive information on FLZ family genes in maize, and shed light on the biological function of ZmFLZ25 in plant ABA signaling.     

Expression of AhATL1, an ABA Transport Factor Gene from Peanut,Is Affected by Altered Memory Gene Expression Patterns and Increased Tolerance to Drought Stress in Arabidopsis

Arachis hypogaea abscisic acid transporter like-1 (AhATL1) modulates abscisic acid (ABA) sensitivity by specifically influencing the importing of ABA into cells, and is a key player in plant stress responses. However, there is limited information on ABA transporters in crops. In this study, we found that the level of AhATL1 expression and AhATL1 distribution increased more rapidly in the second drought (D2) compared with in the first drought (D1). Compared with the first recovery (R1), the AhATL1 expression level and ABA content remained at a higher level during the second recovery (R2). The heterologous overexpression of AhATL1 in Arabidopsis changed the expression pattern of certain memory genes and changed the post response gene type into the memory gene type. Regarding the proline and water content of Col (Arabidopsis thaliana L. Heynh., Col-0), atabcg22, and AhATL1-OX during drought training, the second drought (D2) was more severe than the first drought (D1), which was more conducive to maintaining the cell osmotic balance and resisting drought. In summary, drought stress memory resulted in a rapid increase in the AhATL1 expression and AhATL1 distribution level, and then raised the endogenous ABA content and changed the post response gene type into the memory gene type, which enhanced the drought resistance and recovery ability.     

Microarray Analysis of Transcriptional Responses to Abscisic Acid and Salt Stress in Arabidopsis thaliana

Abscisic acid (ABA) plays a crucial role in plant responses to abiotic stress. To investigate differences in plant responses to salt and ABA stimulus, differences in gene expression in Arabidopsis in response to salt and ABA were compared using an Agilent oligo microarray. A total of 144 and 139 genes were significantly up- and downregulated, respectively, under NaCl stress, while 406 and 381 genes were significantly up- and downregulated, respectively, under ABA stress conditions. In addition, 31 genes were upregulated by both NaCl and ABA stresses, and 23 genes were downregulated by these stressors, suggesting that these genes may play similar roles in plant responses to salt and ABA stress. Gene ontology (GO) analysis revealed four subgroups of genes, including genes in the GO categories “Molecular transducer activity”, “Growth”, “Biological adhesion” and “Pigmentation”, which were expressed in response to ABA stress but not NaCl stress. In addition, genes that play specific roles during salt or ABA stress were identified. Our results may help elucidate differences in the response of plants to salt and ABA stress.