miR394 modulates brassinosteroid signaling to regulate hypocotyl elongation in Arabidopsis.

The interplay between microRNAs (miRNAs) and phytohormones allows plants to integrate multiple internal and external signals to optimize their survival of different environmental conditions. Here, we report that miR394 and its target gene LEAF CURLING RESPONSIVENESS (LCR), which are transcriptionally responsive to BR, participate in BR signaling to regulate hypocotyl elongation in Arabidopsis thaliana. Phenotypic analysis of various transgenic and mutant lines revealed that miR394 negatively regulates BR signaling during hypocotyl elongation, whereas LCR positively regulates this process. Genetically, miR394 functions upstream of BRASSINOSTEROID INSENSITIVE2 (BIN2), BRASSINAZOLEs RESISTANT1 (BZR1), and BRI1-EMS-SUPPRESSOR1 (BES1), but interacts with BRASSINOSTEROID INSENSITIVE1 (BRI1) and BRI1 SUPRESSOR PROTEIN (BSU1). RNA-sequencing analysis suggested that miR394 inhibits BR signaling through BIN2, as miR394 regulates a significant number of genes in common with BIN2. Additionally, miR394 increases the accumulation of BIN2 but decreases the accumulation of BZR1 and BES1, which are phosphorylated by BIN2. MiR394 also represses the transcription of PACLOBUTRAZOL RESISTANCE1/5/6 and EXPANSIN8, key genes that regulate hypocotyl elongation and are targets of BZR1/BES1. These findings reveal a new role for a miRNA in BR signaling in Arabidopsis.

Genome-Wide Identification and Expression Profiling of the ABF Transcription Factor Family in Wheat (Triticum aestivum L.).

Members of the abscisic acid (ABA)-responsive element (ABRE) binding factor (ABF) and ABA-responsive element binding protein (AREB) families play essential roles in the regulation of ABA signaling pathway activity and shape the ability of plants to adapt to a range of stressful environmental conditions. To date, however, systematic genome-wide analyses focused on the ABF/AREB gene family in wheat are lacking. Here, we identified 35 ABF/AREB genes in the wheat genome, designated TaABF1-TaABF35 according to their chromosomal distribution. These genes were further classified, based on their phylogenetic relationships, into three groups (A-C), with the TaABF genes in a given group exhibiting similar motifs and similar numbers of introns/exons. Cis-element analyses of the promoter regions upstream of these TaABFs revealed large numbers of ABREs, with the other predominant elements that were identified differing across these three groups. Patterns of TaABF gene expansion were primarily characterized by allopolyploidization and fragment duplication, with purifying selection having played a significant role in the evolution of this gene family. Further expression profiling indicated that the majority of the TaABF genes from groups A and B were highly expressed in various tissues and upregulated following abiotic stress exposure such as drought, low temperature, low nitrogen, etc., while some of the TaABF genes in group C were specifically expressed in grain tissues. Regulatory network analyses revealed that four of the group A TaABFs (TaABF2, TaABF7, TaABF13, and TaABF19) were centrally located in protein-protein interaction networks, with 13 of these TaABF genes being regulated by 11 known miRNAs, which play important roles in abiotic stress resistance such as drought and salt stress. The two primary upstream transcription factor types found to regulate TaABF gene expression were BBR/BPC and ERF, which have previously been reported to be important in the context of plant abiotic stress responses. Together, these results offer insight into the role that the ABF/AREB genes play in the responses of wheat to abiotic stressors, providing a robust foundation for future functional studies of these genes.

Comparative Transcriptome Analysis Reveals the Genes and Pathways Related to Wheat Root Hair Length.

Tube-like outgrowths from root epidermal cells, known as root hairs, enhance water and nutrient absorption, facilitate microbial interactions, and contribute to plant anchorage by expanding the root surface area. Genetically regulated and strongly influenced by environmental conditions, longer root hairs generally enhance water and nutrient absorption, correlating with increased stress resistance. Wheat, a globally predominant crop pivotal for human nutrition, necessitates the identification of long root hair genotypes and their regulatory genes to enhance nutrient capture and yield potential. This study focused on 261 wheat samples of diverse genotypes during germination, revealing noticeable disparities in the length of the root hair among the genotypes. Notably, two long root hair genotypes (W106 and W136) and two short root hair genotypes (W90 and W100) were identified. Transcriptome sequencing resulted in the development of 12 root cDNA libraries, unveiling 1180 shared differentially expressed genes (DEGs). Further analyses, including GO function annotation, KEGG enrichment, MapMan metabolic pathway analysis, and protein-protein interaction (PPI) network prediction, underscored the upregulation of root hair length regulatory genes in the long root hair genotypes. These included genes are associated with GA and BA hormone signaling pathways, FRS/FRF and bHLH transcription factors, phenylpropanoid, lignin, lignan secondary metabolic pathways, the peroxidase gene for maintaining ROS steady state, and the ankyrin gene with diverse biological functions. This study contributes valuable insights into modulating the length of wheat root hair and identifies candidate genes for the genetic improvement of wheat root traits.

Impact of "Green Revolution" gene Rht-B1b on coleoptile length of wheat.

Wheat coleoptile is a sheath-like structure that helps to deliver the first leaf from embryo to the soil surface. Here, a RIL population consisting of 245 lines derived from Zhou 8425B × Chinese Spring cross was genotyped by the high-density Illumina iSelect 90K assay for coleoptile length (CL) QTL mapping. Three QTL for CL were mapped on chromosomes 2BL, 4BS and 4DS. Of them, two major QTL QCL.qau-4BS and QCL.qau-4DS were detected, which could explain 9.1%-22.2% of the phenotypic variances across environments on Rht-B1 and Rht-D1 loci, respectively. Several studies have reported that Rht-B1b may reduce the length of wheat CL but no study has been carried out at molecular level. In order to verify that the Rht-B1 gene is the functional gene for the 4B QTL, an overexpression line Rht-B1b-OE and a CRISPR/SpCas9 line Rht-B1b-KO were studied. The results showed that Rht-B1b overexpression could reduce the CL, while loss-of-function of Rht-B1b would increase the CL relative to that of the null transgenic plants (TNL). To dissect the underlying regulatory mechanism of Rht-B1b on CL, comparative RNA-Seq was conducted between Rht-B1b-OE and TNL. Transcriptome profiles revealed a few key pathways involving the function of Rht-B1b in coleoptile development, including phytohormones, circadian rhythm and starch and sucrose metabolism. Our findings may facilitate wheat breeding for longer coleoptiles to improve seedling early vigor for better penetration through the soil crust in arid regions.

HY5 inhibits in vitro shoot stem cell niches initiation via directly repressing pluripotency and cytokinin pathways.

The efficiency of plant regeneration from explants is influenced by phytohormones and environmental conditions. Light has a particularly marked effect on in vitro shoot regeneration, and some light signaling factors are involved in shoot regeneration, while the underlying molecular mechanism remains elusive. Here, ELONGATED HYPOCOTYL5 (HY5), as the key transcription factor of light signaling, was found to inhibit shoot regeneration under a range of light conditions. The heightened shoot regeneration capacity of the hy5-215 mutant was less marked in the dark than in the light, showing that HY5-mediated inhibition of shoot regeneration is partly light dependent. The co-localization of WUSCHEL (WUS) and CLAVATA3 (CLV3) expressions was found to coincide with the initiation of stem cell niches in root explants during shoot regeneration. HY5 could directly repress CLV3 and WUS expression by binding to their respective promoters. In parallel, HY5 indirectly repressed CLV3 and WUS by binding to the ARABIDOPSIS RESPONSE REGULATOR12 (ARR12) promoter. The resulting dual regulation exerted by HY5 on WUS and CLV3 impeded the initiation of shoot stem cell niches. A HY5-mediated inhibitory pathway was identified that links cytokinin signaling and the pluripotency pathway during shoot regeneration.

Genome-wide identification of sucrose non-fermenting-1-related protein kinase genes in maize and their responses to abiotic stresses.

Introduction: Protein kinases play an important role in plants in response to environmental changes through signal transduction. As a large family of protein kinases, sucrose non-fermenting-1 (SNF1)-related kinases (SnRKs) were found and functionally verified in many plants. Nevertheless, little is known about the SnRK family of Zea mays. Methods: Evolutionary relationships, chromosome locations, gene structures, conserved motifs, and cis-elements in promoter regions were systematically analyzed. Besides, tissue-specific and stress-induced expression patterns of ZmSnRKs were determined. Finally, functional regulatory networks between ZmSnRKs and other proteins or miRNAs were constructed. Results and Discussion: In total, 60 SnRK genes located on 10 chromosomes were discovered in maize. ZmSnRKs were classified into three subfamilies (ZmSnRK1, ZmSnRK2, and ZmSnRK3), consisting of 4, 14, and 42 genes, respectively. Gene structure analysis showed that 33 of the 42 ZmSnRK3 genes contained only one exon. Most ZmSnRK genes contained at least one ABRE, MBS, and LTR cis-element and a few ZmSnRK genes had AuxRR-core, P-box, MBSI, and SARE ciselements in their promoter regions. The Ka:Ks ratio of 22 paralogous ZmSnRK gene pairs revealed that the ZmSnRK gene family had experienced a purifying selection. Meanwhile, we analyzed the expression profiles of ZmSnRKs, and they exhibited significant differences in various tissues and abiotic stresses. In addition, A total of eight ZmPP2Cs, which can interact with ZmSnRK proteins, and 46 miRNAs, which can target 24 ZmSnRKs, were identified. Generally, these results provide valuable information for further function verification of ZmSnRKs, and improve our understanding of the role of ZmSnRKs in the climate resilience of maize.

Cytosine methylation of the FWA promoter promotes direct in vitro shoot regeneration in Arabidopsis thaliana.

Epigenetic modifications within promoter sequences can act as regulators of gene expression. Shoot regeneration is influenced by both DNA methylation and histone methylation, but the mechanistic basis of this regulation is obscure. Here, we identified 218 genes related to the regeneration capacity of callus that were differentially transcribed between regenerable calli (RC) and non-regenerable calli (NRC) in Arabidopsis thaliana. An analysis of the promoters of five of the differentially expressed genes (FWA, ACC1, TFL1, MAX3, and GRP3) pointed to an inverse relationship between cytosine methylation and transcription. The FWA promoter was demethylated and highly expressed in NRC, whereas it was methylated and expressed at low levels in RC. Explants of the hypomethylation mutants fwa-1 and fwa-2 showed strong levels of FWA expression and regenerated less readily than the wild type, suggesting that FWA inhibits direct in vitro shoot regeneration. WUSCHEL-RELATED HOMEOBOX 9 (WOX9), which is required for shoot apical meristem formation, was directly repressed by FWA. Overexpressing WOX9 partly rescued the shoot regeneration defect of fwa-2 plants. These findings suggest that cytosine methylation of the FWA promoter forms part of the regulatory system governing callus regenerability and direct in vitro shoot regeneration.

Arabidopsis WRKY71 regulates ethylene-mediated leaf senescence by directly activating EIN2, ORE1 and ACS2 genes.

Leaf senescence is a pivotal step in the last stage of the plant life cycle and is influenced by various external and endogenous cues. A series of reports have indicated the involvement of the WRKY transcription factors in regulating leaf senescence, but the molecular mechanisms and signaling pathways remain largely unclear. Here we provide evidence demonstrating that WRKY71 acts as a positive regulator of leaf senescence in Arabidopsis. WRKY71-1D, an overexpressor of WRKY71, exhibited early leaf senescence, while wrky71-1, the WRKY71 loss-of-function mutant, displayed delayed leaf senescence. Accordingly, a set of senescence-associated genes (SAGs) were substantially elevated in WRKY71-1D but markedly decreased in wrky71-1. Chromatin immunoprecipitation assays indicated that WRKY71 can bind directly to the promoters of SAG13 and SAG201. Transcriptome analysis suggested that WRKY71 might mediate multiple cues to accelerate leaf senescence, such as abiotic stresses, dark and ethylene. WRKY71 was ethylene inducible, and treatment with the ethylene precursor 1-amino-cyclopropane-1-carboxylic acid enhanced leaf senescence in WRKY71-1D but caused only a marginal delay in leaf senescence in wrky71-1. In vitro and in vivo assays demonstrated that WRKY71 can directly regulate ETHYLENE INSENSITIVE2 (EIN2) and ORESARA1 (ORE1), genes of the ethylene signaling pathway. Consistently, leaf senescence of WRKY71-1D was obviously retarded in the ein2-5 and nac2-1 mutants. Moreover, WRKY71 was also proved to interact with ACS2 in vitro and in vivo. Treatment with AgNO3 and aminoethoxyvinylglycine and acs2-1 could greatly arrest the leaf senescence of WRKY71-1D. In conclusion, our data revealed that WRKY71 mediates ethylene signaling and synthesis to hasten leaf senescence in Arabidopsis.

MiR160 and its target genes ARF10, ARF16 and ARF17 modulate hypocotyl elongation in a light, BRZ, or PAC-dependent manner in Arabidopsis: miR160 promotes hypocotyl elongation.

Multiple hormonal and environmental signals participate in the regulation of plant hypocotyl elongation, which allow the plants to optimize their survival strategy from seed germination to seedling establishment. Auxin plays key roles in cell elongation via auxin signaling transduction and its interactions with other hormonal and environmental signals. However, the roles of auxin response factor (ARF) family in cross-talk between auxin and other hormonal or environmental signals during hypocotyl elongation are not fully understood. Here we show that miR160 and its target genes ARF10, ARF16 and ARF17 modulate hypocotyl elongation in a light, brassinazole (BRZ, a BR biosynthesis inhibitor), or paclobutrazol (PAC, a GA biosynthesis inhibitor)-dependent manner in Arabidopsis. miR160, ARF10, ARF16 and ARF17 have no effects on hypocotyl elongation in the dark. However, in the presence of either light, BRZ, or PAC, ARF10, ARF16 and ARF17 inhibit hypocotyl elongation, and miR160 promotes hypocotyl elongation via cleavage of their mRNA. miR160 and ARF10 are both expressed in the hypocotyl. ARF10 represses the expression of PACLOBUTRAZOL RESISTANCE1 (PRE1) and 35S::PRE1 could partly rescue the phenotype of mARF10 (a miR160-resistant form of ARF10), suggesting that PRE1 acts downstream of ARF10 in regulating hypocotyl elongation. In conclusion, our results indicate that miR160-ARF10/16/17 might serve as a molecular link in cross-talk of auxin, light, BR, and GA in hypocotyl elongation.

The Type-B Cytokinin Response Regulator ARR1 Inhibits Shoot Regeneration in an ARR12-Dependent Manner in Arabidopsis.

Exogenous cytokinin is critical for in vitro shoot regeneration. Proteins involved in the cytokinin signal transduction pathway, including type-B ARABIDOPSIS RESPONSE REGULATORs (ARRs), participate in shoot regeneration in Arabidopsis (Arabidopsis thaliana). Some type-B ARRs (e.g., ARR1 and ARR12) promote shoot regeneration by directly activating WUSCHEL (WUS) expression; however, it is unclear how type-B ARRs inhibit shoot regeneration. Here, we show that ARR12 is a central enhancer of callus formation and shoot regeneration, whereas ARR1 is a strong inhibitor of this process that counteracts the positive effect of ARR12. ARR1 indirectly represses CLAVATA3 (CLV3) expression in an ARR12-dependent manner via competing with ARR12 for binding to the CLV3 promoter, which contributes to its ARR12-dependent inhibitory effect on callus formation and shoot regeneration. In parallel, ARR1 inhibits shoot regeneration through transcriptional activation of INDOLE-3-ACETIC ACID INDUCIBLE17, an auxin response repressor gene, and the consequent indirect repression of WUS expression. Thus, type-B ARRs have diverse effects on callus formation and shoot regeneration. Our study reveals novel molecular pathways linking cytokinin signaling, the CLV3 regulator, and auxin signaling, and sheds light on the mechanism underlying cytokinin-regulated shoot regeneration.

MYB94 and MYB96 additively inhibit callus formation via directly repressing LBD29 expression in Arabidopsis thaliana.

Plant somatic cells can be reprogrammed during in vitro culture. Callus induction is the initial step of a typical plant regeneration system. Recent studies showed that auxin-induced callus formation in multiple organs occurs from the pericycle or pericycle-like cells via a root developmental pathway. However, the molecular control of callus formation is largely unknown. Here, two MYB transcription factors, MYB94 and MYB96, were shown to play negative roles in auxin-induced callus formation in Arabidopsis. MYB94 and MYB96 were expressed in the newly formed callus. myb96, myb94, and myb94 myb96 generated more calli than the WT, with myb94 myb96 producing the most. MYB94 and MYB96 repressed expression of LATERAL ORGAN BOUNDARIES-DOMAIN 29 (LBD29) via directly binding to the gene's promoter. The loss of function of LBD29 partly rescued the callus formation defect of myb94 myb96. Our findings found MYB94 and MYB96 to be important repressors of callus formation and MYB94/96-LBD29 as a new regulatory pathway acting in parallel with ARF7/19-LBDs' pathway to modulate in vitro callus formation.

ARGONAUTE10 Inhibits In Vitro Shoot Regeneration Via Repression of miR165/166 in Arabidopsis thaliana.

Many plant cells retain their totipotency when cultured in vitro. The regulation of shoot regeneration from in vitro culture involves a number of gene products, but the nature of the associated post-transcriptional events remains largely unknown. Here, the post-transcriptional regulator ARGONAUTE10 (AGO10), a protein which is specifically expressed in the explant during the period when pro-shoot apical meristems (SAMs) are forming, has been known to inhibit shoot regeneration. In in vitro cultured explants of the loss-of-function mutant ago10, a much larger than normal number of SAMs was formed and, in these, the stem cell marker genes WUSCHEL, CLAVATA3 and SHOOT MERISTEMLESS were all strongly expressed. AGO10 repressed the accumulation of the microRNAs miR165/166, thereby up-regulating a suite of HD-ZIP III genes. The overproduction of miR166 was shown to promote shoot regeneration, while the absence of miR165/166 message resulted in a blockage to shoot regeneration and only a partial rescue of the phenotype of the ago10 mutant. The major conclusion was that the shoot regeneration inhibition determined by AGO10 functions via the repression of miR165/166.

Primary root growth in Arabidopsis thaliana is inhibited by the miR159 mediated repression of MYB33, MYB65 and MYB101.

Organ growth is a fundamental developmental process basing on cell proliferation and differentiation. The growth of the plant root is sustained by the activity of the root meristem, a process controlled in part by various transcription factors. Here, the miR159 has been identified as a post transcriptional repressor of root growth, on the basis that the mir159ab double mutant developed a larger meristem than did the wild type, and that it formed longer roots. In the mutant, the abundance of MYB33, MYB65 and MYB101 transcript was substantially increased. When MYB33, MYB65 and MYB101 were replaced by the miR159-resistant forms mMYB33, mMYB65 and mMYB101 respectively, the root meristem was similarly enlarged and the growth of the primary root enhanced. MYB65 activity promoted cell division in the root meristem by accelerating the cell cycle. The data suggest that miR159 acts as a key repressor of the primary root's growth, acting through its repression of MYB65 and consequent blocking of the cell cycle.

ARR12 promotes de novo shoot regeneration in Arabidopsis thaliana via activation of WUSCHEL expression.

Auxin and cytokinin direct cell proliferation and differentiation during the in vitro culture of plant cells, but the molecular basis of these processes, especially de novo shoot regeneration, has not been fully elucidated. Here, we describe the regulatory control of shoot regeneration in Arabidopsis thaliana (L.) Heynh, based on the interaction of ARABIDOPSIS RESPONSE REGULATOR12 (ARR12) and WUSCHEL (WUS). The major site of ARR12 expression coincided with the location where the shoot apical meristem (SAM) initiated. The arr12 mutants showed severely impaired shoot regeneration and reduced responsiveness to cytokinin; consistent with this, the overexpression of ARR12 enhanced shoot regeneration. Certain shoot meristem specification genes, notably WUSCHEL (WUS) and CLAVATA3, were significantly downregulated in the arr12 explants. Chromatin immunoprecipitation (ChIP) and transient activation assays demonstrated that ARR12 binds to the promoter of WUS. These observations indicate that during shoot regeneration, in vitro, ARR12 functions as a molecular link between cytokinin signaling and the expression of shoot meristem specification genes.