Genome-Wide cis-Regulatory Element Based Discovery of Auxin-Responsive Genes in Higher Plant

Auxin has a profound impact on plant physiology and participates in almost all aspects of plant development processes. Auxin exerts profound pleiotropic effects on plant growth and differentiation by regulating the auxin response genes’ expressions. The classical auxin reaction is usually mediated by auxin response factors (ARFs), which bind to the auxin response element (AuxRE) in the promoter region of the target gene. Experiments have generated only a limited number of plant genes with well-characterized functions. It is still unknown how many genes respond to exogenous auxin treatment. An economical and effective method was proposed for the genome-wide discovery of genes responsive to auxin in a model plant, Arabidopsis thaliana (A. thaliana). Our method relies on cis-regulatory-element-based targeted gene finding across different promoters in a genome. We first exploit and analyze auxin-specific cis-regulatory elements for the transcription of the target genes, and then identify putative auxin responsive genes whose promoters contain the elements in the collection of over 25,800 promoters in the A. thaliana genome. Evaluating our result by comparing with a published database and the literature, we found that this method has an accuracy rate of 65.2% (309/474) for predicting candidate genes responsive to auxin. Chromosome distribution and annotation of the putative auxin-responsive genes predicted here were also mined. The results can markedly decrease the number of identified but merely potential auxin target genes and also provide useful clues for improving the annotation of gene that lack functional information.

The FBA Motif-Containing Protein NpFBA1 Causes Leaf Curling and Reduces Resistance to Black Shank Disease in Tobacco

Plant leaf morphology has a great impact on plant drought resistance, ornamental research and leaf yield. In this study, we identified a new gene in Nicotiana plumbaginifolia, NpFBA1, that causes leaf curl. The results show that the NpFBA1 protein contains only one unique F-box associated (FBA) domain and does not have an F-box conserved domain. Phylogenetic analysis placed this gene and other Nicotiana FBA genes on a separate branch, and the NpFBA1 protein localized to the nucleus and cytoplasm. The expression of NpFBA1 was induced by black shank pathogen (Phytophthora parasitica var. nicotianae) infection and treatment with salicylic acid (SA) and methyl jasmonate (MeJA). NpFBA1-overexpressing transgenic lines showed leaf curling and aging during the rosette phase. During the bolting period, the leaves were curly and rounded, and the plants were dwarfed. In addition, NpFBA1-overexpressing lines were more susceptible to disease than wild-type (WT) plants. Further studies revealed that overexpression of NpFBA1 significantly downregulated the expression of auxin response factors such as NtARF3 and the lignin synthesis genes NtPAL, NtC4H, NtCAD2, and NtCCR1 in the leaves. In conclusion, NpFBA1 may play a key role in regulating leaf development and the response to pathogen infection.

Dual Catalytic Hairpin Assembly-Based Automatic Molecule Machine for Amplified Detection of Auxin Response Factor-Targeted MicroRNA-160

MicroRNA160 plays a crucial role in plant development by negatively regulating the auxin response factors (ARFs). In this manuscript, we design an automatic molecule machine (AMM) based on the dual catalytic hairpin assembly (D-CHA) strategy for the signal amplification detection of miRNA160. The detection system contains four hairpin-shaped DNA probes (HP1, HP2, HP3, and HP4). For HP1, the loop is designed to be complementary to miRNA160. A fragment of DNA with the same sequences as miRNA160 is separated into two pieces that are connected at the 3′ end of HP2 and 5′ end of HP3, respectively. In the presence of the target, four HPs are successively dissolved by the first catalytic hairpin assembly (CHA1), forming a four-way DNA junction (F-DJ) that enables the rearrangement of separated DNA fragments at the end of HP2 and HP3 and serving as an integrated target analogue for initiating the second CHA reaction, generating an enhanced fluorescence signal. Assay experiments demonstrate that D-CHA has a better performance compared with traditional CHA, achieving the detection limit as low as 10 pM for miRNA160 as deduced from its corresponding DNA surrogates. Moreover, non-target miRNAs, as well as single-base mutation targets, can be detected. Overall, the D-CHA strategy provides a competitive method for plant miRNAs detection.

Transcriptome Analysis of Lolium temulentum Exposed to a Combination of Drought and Heat Stress

Drought and heat are two major stresses predicted to increase in the future due to climate change. Plants exposed to multiple stressors elicit unique responses from those observed under individual stresses. A comparative transcriptome analysis of Lolium temulentum exposed to drought plus heat and non-stressed control plants revealed 20,221 unique up-regulated and 17,034 unique down-regulated differentially regulated transcripts. Gene ontology analysis revealed a strong emphasis on transcriptional regulation, protein folding, cell cycle/parts, organelles, binding, transport, signaling, oxidoreductase, and antioxidant activity. Differentially expressed genes (DEGs) encoding for transcriptional control proteins such as basic leucine zipper, APETALA2/Ethylene Responsive Factor, NAC, and WRKY transcription factors, and Zinc Finger (CCCH type and others) proteins were more often up-regulated, while DEGs encoding Basic Helix-Loop-Helix, MYB and GATA transcription factors, and C2H2 type Zinc Finger proteins were more often down-regulated. The DEGs encoding heat shock transcription factors were only up-regulated. Of the hormones, auxin-related DEGs were the most prevalent, encoding for auxin response factors, binding proteins, and efflux/influx carriers. Gibberellin-, cytokinin- and ABA-related DEGs were also prevalent, with fewer DEGs related to jasmonates and brassinosteroids. Knowledge of genes/pathways that grasses use to respond to the combination of heat/drought will be useful in developing multi-stress resistant grasses.

Genome-wide identification and expression analysis of auxin response factors in peanut (Arachis hypogaea L.)

Auxin response factors (ARFs) are transcription factors that regulate the expression of auxin response genes, and have important functions in plant growth and development. In this study, available genome data for peanut (Arachis hypogaea L.) were used to identify AhARF genes. In total, 61 AhARFs and 23 AtARFs were divided into six groups (I–VI). Molecular structural analysis revealed that the protein members of AhARF contain at least two domains, the B3 domain and the Auxin-resp domain, and that some have a C-terminal dimerisation domain. Screening of the transcriptome data of 22 tissues of A. hypogaea cv. Tifrunner in a public database showed high expression levels of AhARF2 and AhARF6. AhARF6 was expressed more highly in the stem and branch than in the root and leaf of the wild species Arachis monticola (A. mon) and cultivated species H103. After treatment with exogenous auxin (NAA), the expression of AhARF6 was inhibited, and this inhibition was greater in A. mon than in H103. The transcriptome map revealed that the expression of AhARF6 was higher in the larger pods of H8107 and ZP06 than in the medium pods of H103 and small pods of A. mon. Moreover, AhARF6-5 was proven to be localised in the nucleus, consistent with the location of AtARF6. These results suggest that AhARF6 may play an important role in pod development in peanut.

Interspecies Evolution and Networks Investigation of the Auxin Response Protein (AUX/IAA) Family Reveals the Adaptation Mechanisms of Halophytes Crops in Nitrogen Starvation Agroecological Environments

The maintenance of adaptability to the exposure to agroecological extreme environments is generally a feature after the long-term domestication of crops. Auxin influences plant growth in all environments. At present, the research on the auxin response factors (ARFs) has been very in-depth. However, there is still a large gap in the research on the origin, evolution, and regulatory networks of the Auxin-responsive protein (AUX/IAA) family. Here, we identified 495 AUX/IAAs from 19 representative species covering aquatic algae to angiosperms and found that they originated from early bryophytes and mainly expanded by polyploidy in angiosperms. In the domesticated crop quinoa, the evolutionary model of the IAA family is relatively independent and forms a robust regulatory network with auxin signals and energy metabolism pathways. In the nitrogen-deficient environment, CqIAAs (Chenopodium quinoa AUX/IAAs), auxin signals, and TCA pathway genes induced expression in young roots to promote root elongation and could regulate the balance of carbon and nitrogen metabolism to maintain the adaptation of early seedlings in poor environments. Furthermore, a rapidly evolving CqIAA (AUR62011942) not only has different expression levels in two quinoa seeds but also has a significant stress response when seedlings face nitrogen deficiency stress, which may be a key factor in the adaptive regulation of the barren environment. Our research provides valuable clues for understanding the origin, evolution, and functional innovation of auxin signaling and also provides a reference for future agricultural breeding in the context of global environmental changes.

Association Analysis Revealed That TaPYL4 Genes Are Linked to Plant Growth Related Traits in Multiple Environment

Abscisic acid (ABA), one of phytohormones, plays an important regulatory role in plant growth and development. ABA receptor PYL4 (pyrabactin resistance 1-like 4) was previously detected to be involved in plant response to a variety of stresses. TaPYL4 overexpression could enhance wheat (Triticum aestivum) drought resistance. In order to further investigate TaPYL4’s role in regulating development of other major agronomic traits in wheat, genes of TaPYL4-2A, TaPYL4-2B, and TaPYL4-2D were cloned from wheat, respectively. Polymorphism analysis on TaPYL4 sequences revealed that encoding regions of the three genes were highly conserved, without any SNP (single nucleotide polymorphism) presence. However, nine SNPs and four SNPs were identified in the promoter regions of TaPYL4-2A and TaPYL4-2B, respectively. Functional molecular markers were developed based on these polymorphisms, which were then used to scan a natural population of 323 common wheat accessions for correlation analysis between genotype and the target phenotypic traits. Both TaPYL4-2A and TaPYL4-2B markers were significantly correlated with plant growth-related traits under multiple environments (well-watered, drought and heat stress treatments). The additive effects of TaPYL4-2A and TaPYL4-2B were verified by the combinational haplotype (Hap-AB1∼Hap-AB4) effects determined from field data. Cis-acting elements were analyzed in the promoters of TaPYL4-2A and TaPYL4-2B, showing that a TGA-element bound by ARFs (auxin response factors) existed only in Hap-2A-1 of TaPYL4-2A. Gene expression assays indicated that TaPYL4-2A was constitutively expressed in various tissues, with higher expression in Hap-2A-1 genotypes than in Hap-2A-2 materials. Notably, TaARF4 could act as TaPYL4-2A transcription activator in Hap-2A-1 materials, but not in Hap-2A-2 genotypes. Analysis of geographic distribution and temporal frequency of haplotypes indicated that Hap-AB1 was positively selected in wheat breeding in China. Therefore, TaPYL4-2A and TaPYL4-2B could be a valuable target gene in wheat genetic improvement to develop the ideal plant architecture.

Genome-Wide Identification of the ARF Gene Family and ARF3 Target Genes Regulating Ovary Initiation in Hazel via ChIP Sequencing

Hazel (Corylus spp.) is an economically important nut species with a unique biological characteristic of ovary differentiation and development initiating from the ovary primordium after pollination. Auxin participates in ovary initiation and has an essential impact on hazel fruit yield and quality. The regulation of auxin in ovary development is thought to be related to auxin response factors (ARFs); however, its detailed regulatory mechanism remains unclear. The spatiotemporal expression pattern of C. heterophylla ARF3 (ChARF3) was accessed via ARF gene family member identification and expression abundance analysis as well as immunohistochemistry. ChARF3 target genes were identified via chromatin immunoprecipitation followed by next-generation sequencing (ChIP-Seq). In total, 14 ChARF members containing at least B3 and Auxin_resp domains were found to be distributed on 9 of 11 chromosomes, and the protein molecular weights were predicted to range from 70.93–139.22 kD. Among eight differentially expressed ChARFs, ChARF3 showed the most significant differences over four ovary developmental stages. Immunohistochemical analysis revealed that ChARF3 was expressed in the ovary primordium and funiculus, integument, endosperm, radicle, and cotyledon indicating its potential regulatory roles in ovary differentiation and development. In total, 3,167 ChARF3 target genes were identified through ChIP-Seq in four ovary developmental stages and were significantly enriched in the biosynthesis of secondary metabolites (ko01110), phenylpropanoid biosynthesis (ko00940), and phytohormone signal transduction (ko04075). ChARF3 was hypothesized to be involved in the regulation of auxin-induced genes and the transcription factors MADS, AP2/ERF, TCP, FT, and LFY. These results suggest that ChARF3 may regulate ovary initiation and ovule development by mediating genes related to auxin biosynthesis and transport, cell division and proliferation, and flower and fruit development. This study provides new insights into the molecular mechanism of hazel yield formation.