Positional cloning and characterization reveal the role of TaSRN-3D and TaBSR1 in the regulation of seminal root number in wheat.

Seminal roots play a critical role in water and nutrient absorption, particularly in the early developmental stages of wheat. However, the genes responsible for controlling SRN in wheat remain largely unknown. Genetic mapping and functional analyses identified a candidate gene (TraesCS3D01G137200, TaSRN-3D) encoding a Ser/Thr kinase glycogen synthase kinase 3 (STKc_GSK3) that regulated SRN in wheat. Additionally, experiments involving hormone treatment, nitrate absorption and protein interaction were conducted to explore the regulatory mechanism of TaSRN-3D. Results showed that the TaSRN-3D4332 allele inhibited seminal roots initiation and development, while loss-of-function mutants showed significantly higher seminal root number (SRN). Exogenous application of epi-brassinolide could increase the SRN in a HS2-allelic background. Furthermore, chlorate sensitivity and 15N uptake assays revealed that a higher number of seminal roots promoted nitrate accumulation. TaBSR1 (BIN2-related SRN Regulator 1, orthologous to OsGRF4/GL2 in rice) acts as an interactor of TaSRN-3D and promotes TaBSR1 degradation to reduce SRN. This study provides valuable insights into understanding the genetic basis and regulatory network of SRN in wheat, highlighting their roles as potential targets for root-based improvement in wheat breeding.

Reshaped DNA methylation cooperating with homoeolog-divergent expression promotes improved root traits in synthesized tetraploid wheat.

Polyploidization is a major event driving plant evolution and domestication. However, how reshaped epigenetic modifications coordinate gene transcription to generate phenotypic variations during wheat polyploidization is currently elusive. Here, we profiled transcriptomes and DNA methylomes of two diploid wheat accessions (SlSl and AA) and their synthetic allotetraploid wheat line (SlSlAA), which displayed elongated root hair and improved root capability for nitrate uptake and assimilation after tetraploidization. Globally decreased DNA methylation levels with a reduced difference between subgenomes were observed in the roots of SlSlAA. DNA methylation changes in first exon showed strong connections with altered transcription during tetraploidization. Homoeolog-specific transcription was associated with biased DNA methylation as shaped by homoeologous sequence variation. The hypomethylated promoters showed significantly enriched binding sites for MYB, which may affect gene transcription in response to root hair growth. Two master regulators in root hair elongation pathway, AlCPC and TuRSL4, exhibited upregulated transcription levels accompanied by hypomethylation in promoter, which may contribute to the elongated root hair. The upregulated nitrate transporter genes, including NPFs and NRTs, also are significantly associated with hypomethylation, indicating an epigenetic-incorporated regulation manner in improving nitrogen use efficiency. Collectively, these results provided new insights into epigenetic changes in response to crop polyploidization and underscored the importance of epigenetic regulation in improving crop traits.

TaNAM-6A is essential for nitrogen remobilisation and regulates grain protein content in wheat (Triticum aestivum L.).

Grain protein content (GPC) is a crucial quality trait in bread wheat, which is influenced by the key transcription factor TaNAM. However, the regulatory mechanisms of TaNAM have remained largely elusive. In this study, a new role of TaNAM was unveiled in regulating nitrogen remobilisation which impacts GPC. The TaNAM knockout mutants generated by clustered regularly interspaced short palindromic repeats/Cas9 exhibited significantly delayed senescence and lower GPC, while overexpression of TaNAM-6A resulted in premature senility and much higher GPC. Further analysis revealed that TaNAM directly activates the genes TaNRT1.1 and TaNPF5.5s, which are involved in nitrogen remobilisation. This activity aids in the transfer of nitrogen from leaves to grains for protein synthesis. In addition, an elite allele of TaNAM-6A, associated with high GPC, was identified as a candidate gene for breeding high-quality wheat. Overall, our work not only elucidates the potential mechanism of TaNAM-6A affecting bread wheat GPC, but also highlights the significance of nitrogen remobilisation from senescent leaves to grains for protein accumulation. Moreover, our research provides a new target and approach for improving the quality traits of wheat, particularly the GPC.

Positional cloning and characterization reveal the role of a miRNA precursor gene ZmLRT in the regulation of lateral root number and drought tolerance in maize.

Lateral roots play essential roles in drought tolerance in maize (Zea mays L.). However, the genetic basis for the variation in the number of lateral roots in maize remains elusive. Here, we identified a major quantitative trait locus (QTL), qLRT5-1, controlling lateral root number using a recombinant inbred population from a cross between the maize lines Zong3 (with many lateral roots) and 87-1 (with few lateral roots). Fine-mapping and functional analysis determined that the candidate gene for qLRT5-1, ZmLRT, expresses the primary transcript for the microRNA miR166a. ZmLRT was highly expressed in root tips and lateral root primordia, and knockout and overexpression of ZmLRT increased and decreased lateral root number, respectively. Compared with 87-1, the ZmLRT gene model of Zong3 lacked the second and third exons and contained a 14 bp deletion at the junction between the first exon and intron, which altered the splicing site. In addition, ZmLRT expression was significantly lower in Zong3 than in 87-1, which might be attributed to the insertions of a transposon and over large DNA fragments in the Zong3 ZmLRT promoter region. These mutations decreased the abundance of mature miR166a in Zong3, resulting in increased lateral roots at the seedling stage. Furthermore, miR166a post-transcriptionally repressed five development-related class-III homeodomain-leucine zipper genes. Moreover, knockout of ZmLRT enhanced drought tolerance of maize seedlings. Our study furthers our understanding of the genetic basis of lateral root number variation in maize and highlights ZmLRT as a target for improving drought tolerance in maize.

A kelch-repeat superfamily gene, ZmNL4, controls leaf width in maize (Zea mays L.).

Leaf width (LW) is an important component of plant architecture that extensively affects both light capture during photosynthesis and grain yield, particularly under dense planting conditions. However, the genetic and molecular mechanisms regulating LW remain largely elusive in maize (Zea mays L.). In this study, qLW4a, a major quantitative trait locus controlling LW, was identified in a population constructed with maize inbred lines PH6WC, with wide leaves, and Lin387, with narrow leaves. Map-based cloning revealed that ZmNL4, a kelch-repeat superfamily gene, emerged to be the candidate for qLW4a, and a single-base deletion in the conserved SMC_prok_B domain of ZmNL4 in Lin387 caused a frame shift, leading to premature termination. Consistently, the knockout of ZmNL4 by CRISPR/Cas9 editing significantly reduced the LW, which was attributed to a reduction in the cell number instead of cell size, indicating a role of ZmNL4 in regulating cell division. Transcriptomic comparison of ZmNL4 knockout lines with the wild type B73-329 revealed that ZmNL4 might participate in cell wall biogenesis, asymmetric cell division, metabolic processes, transmembrane transport and response to external stimulus, etc. These results provide insights into the genetic and molecular mechanisms of ZmNL4 in controlling LW and could potentially contribute to optimizing plant architecture for maize breeding.

A single nucleotide deletion in the third exon of FT-D1 increases the spikelet number and delays heading date in wheat (Triticum aestivum L.).

The spikelet number and heading date are two crucial and correlated traits for yield in wheat. Here, a quantitative trait locus (QTL) analysis was conducted in F8 recombinant inbred lines (RILs) derived from crossing two common wheats with different spikelet numbers. A total of 15 stable QTL influencing total spikelet number (TSN) and heading date (HD) were detected. Notably, FT-D1, a well-known flowering time gene in wheat, was located within the finely mapped interval of a major QTL on 7DS (QTsn/Hd.cau-7D). A causal indel of one G in the third exon of FT-D1 was significantly associated with total spikelet number and heading date. Consistently, CRISPR/Cas9 mutant lines with homozygous mutations in FT-D1 displayed an increase in total spikelet number and heading date when compared with wild type. Moreover, one simple and robust marker developed according to the polymorphic site of FT-D1 revealed that this one G indel had been preferentially selected to adapt to different environments. Collectively, these data provide further insights into the genetic basis of spikelet number and heading date, and the diagnostic marker of FT-D1 will be useful for marker-assisted pyramiding in wheat breeding.

Heat Stress Tolerance 2 confers basal heat stress tolerance in allohexaploid wheat (Triticum aestivum L.).

Heat stress substantially reduces the yield potential of wheat (Triticum aestivum L.), one of the most widely cultivated staple crops, and greatly threatens global food security in the context of global warming. However, few studies have explored the heat stress tolerance (HST)-related genetic resources in wheat. Here, we identified and fine-mapped a wheat HST locus, TaHST2, which is indispensable for HST in both the vegetative and reproductive stages of the wheat life cycle. The studied pair of near isogenic lines (NILs) exhibited diverse morphologies under heat stress, based on which we mapped TaHST2 to a 485 kb interval on chromosome arm 4DS. Under heat stress, TaHST2 confers a superior conversion rate from soluble sugars to starch in wheat grains, resulting in faster grain filling and a higher yield potential. A further exploration of genetic resources indicated that TaHST2 underwent strong artificial selection during wheat domestication, suggesting it is an essential locus for basal HST in wheat. Our findings provide deeper insights into the genetic basis of wheat HST and might be useful for global efforts to breed heat-stress-tolerant cultivars.

Identification and characterization of QTL for spike morphological traits, plant height and heading date derived from the D genome of natural and resynthetic allohexaploid wheat.

KEY MESSAGE: QHd.cau-7D.1 for heading date was delimited into the physical interval of approximately 17.38 Mb harboring three CONSTANS-like zinc finger genes. Spike morphological traits, plant height and heading date play important roles in yield improvement of wheat. To reveal the genetic factors that controlling spike morphological traits, plant height and heading date on the D genome, we conducted analysis of quantitative traits locus (QTL) using 198 F7:8 recombinant inbred lines (RILs) derived from a cross between the common wheat TAA10 and resynthesized allohexaploid wheat XX329 with similar AABB genomes. A total of 23 environmentally stable QTL on the D sub-genome for spike length (SL), fertile spikelet number per spike (FSN), sterile spikelet number per spike (SSN), total spikelet number per spike (TSN), spike compactness (SC), plant height (PHT) and heading date (HD) were detected, among which eight appeared to be novel QTL. Furthermore, QHd.cau-7D.1 and QPht.cau-7D.2 shared identical confidence interval and were delimited into the physical interval of approximately 17.38 Mb with 145 annotated genes, including three CONSTANS-like zinc finger genes (TraesCS7D02G209000, TraesCS7D02G213000 and TraesCS7D02G220300). This study will help elucidate the molecular mechanism of the seven traits (SL, FSN, SSN, TSN, SC, PHT and HD) and provide a potentially valuable resource for genetic improvement.

FRIZZY PANICLE defines a regulatory hub for simultaneously controlling spikelet formation and awn elongation in bread wheat.

Grain yield in bread wheat (Triticum aestivum L.) is largely determined by inflorescence architecture. Zang734 is an endemic Tibetan wheat variety that exhibits a rare triple spikelet (TRS) phenotype with significantly increased spikelet/floret number per spike. However, the molecular basis underlying this specific spike morphology is completely unknown. Through map-based cloning, the causal genes for TRS trait in Zang734 were isolated. Furthermore, using CRISPR/Cas9-based gene mutation, transcriptome sequencing and protein-protein interaction, the downstream signalling networks related to spikelet formation and awn elongation were defined. Results showed that the null mutation in WFZP-A together with deletion of WFZP-D led to the TRS trait in Zang734. More interestingly, WFZP plays a dual role in simultaneously repressing spikelet formation gene TaBA1 and activating awn development genes, basically through the recruitments of chromatin remodelling elements and the Mediator complex. Our findings provide insights into the molecular bases by which WFZP suppresses spikelet formation but promotes awn elongation and, more importantly, define WFZP-D as a favourable gene for high-yield crop breeding.

A C-terminal encoded peptide, ZmCEP1, is essential for kernel development in maize.

C-terminal encoded peptides (CEPs) are peptide hormones which act as mobile signals coordinating important developmental programs. Previous studies have unraveled that CEPs are able to regulate plant growth and abiotic stress via cell-to-cell communication in Arabidopsis and rice; however, little is known about their roles in maize. Here, we examined the spatiotemporal expression pattern of ZmCEP1 and showed that ZmCEP1 is highly expressed in young ears and tassels of maize, particularly in the vascular bundles of ears. Heterologous expression of ZmCEP1 in Arabidopsis results in smaller plants and seed size. Similarly, overexpression of ZmCEP1 in maize decreased the plant and ear height, ear length, kernel size, and 100-kernel weight. Consistently, exogenous application of the synthesized ZmCEP1 peptide to the roots of Arabidopsis and maize inhibited root elongation. Knock-out of ZmCEP1 through CRISPR/Cas9 significantly increased plant and ear height, kernel size and 100-kernel weight. Transcriptome analysis revealed that knock-out of ZmCEP1 up-regulated a subset of genes involved in nitrogen metabolism, nitrate transport, sugar transport and auxin response. Thus, these results provide new insights into the genetic and molecular function of ZmCEP1 in regulating kernel development and plant growth, providing novel opportunities for maize breeding.

The decreased expression of GW2 homologous genes contributed to the increased grain width and thousand­grain weight in wheat-Dasypyrum villosum 6VS·6DL translocation lines.

KEY MESSAGE: This study demonstrated that the aberrant transcription of DvGW2 contributed to the increased grain width and thousand-grain weight in wheat-Dasypyrum villosum T6VS·6DL translocation lines. Due to the high immunity to powdery mildew, Dasypyrum villosum 6VS has been one of the most successful applications of the wild relatives in modern wheat breeding. Along with the desired traits, side-effects could be brought when large alien chromosome fragments are introduced into wheat, but little is known about effects of 6VS on agronomic traits. Here, we found that T6VS·6DL translocation had significantly positive effects on grain weight, plant heightand spike length, and small negative effects on total spikelet number and spikelet compactness using recipient and wheat-D. villosum T6VS·6DL allohexaploid wheats, Wan7107 and Pm97033. Further analysis showed that the 6VS segment might exert direct genetic effect on grain width, then driving the increase of thousand-grain weight. Furthermore, comparative transcriptome analysis identified 2549 and 1282 differentially expressed genes (DEGs) and 2220 and 1496 specifically expressed genes (SEGs) at 6 days after pollination (DAP) grains and 15 DAP endosperms, respectively. Enrichment analysis indicated that the process of cell proliferation category was over-represented in the DEGs. Notably, two homologous genes, TaGW2-D1 and DvGW2, were identified as putative candidate genes associated with grain weight and yield. The expression analysis showed that DvGW2 had an aberrant expression in Pm97033, resulting in significantly lower total expression level of GW2 than Wan7107, which drives the increase of grain weight and width in Pm97033. Collectively, our data indicated that the compromised expression of DvGW2 is critical for increased grain width and weight in T6VS·6DL translocation lines.

Histone acetyltransferase GCN5-mediated regulation of long non-coding RNA At4 contributes to phosphate starvation response in Arabidopsis.

Phosphate availability is becoming a limiting environmental factor that inhibits plant growth and development. Here, we demonstrated that mutation of the histone acetyltransferase GCN5 impaired phosphate starvation responses (PSRs) in Arabidopsis. Transcriptome analysis revealed that 888 GCN5-regulated candidate genes were potentially involved in responding to phosphate starvation. ChIP assay indicated that four genes, including a long non-coding RNA (lncRNA) At4, are direct targets of GCN5 in PSR regulation. In addition, GCN5-mediated H3K9/14 acetylation of At4 determined dynamic At4 expression. Consistent with the function of At4 in phosphate distribution, mutation of GCN5 impaired phosphate accumulation between shoots and roots under phosphate deficiency condition, whereas constitutive expression of At4 in gcn5 mutants partially restored phosphate relocation. Further evidence proved that GCN5 regulation of At4 influenced the miRNA miR399 and its target PHO2 mRNA level. Taken together, we propose that GCN5-mediated histone acetylation plays a crucial role in PSR regulation via the At4-miR399-PHO2 pathway and provides a new epigenetic mechanism for the regulation of lncRNA in plants.

The E3 Ligase TaSAP5 Alters Drought Stress Responses by Promoting the Degradation of DRIP Proteins.

In Arabidopsis (Arabidopsis thaliana) plants growing under normal conditions, DEHYDRATION-RESPONSIVE ELEMENT BINDING PROTEIN2A (DREB2A) is present at low levels because it is ubiquitinated and destabilized by DREB2A INTERACTING PROTEIN1 (DRIP1) and DRIP2 through 26S proteasome-mediated proteolysis. Drought stress counteracts the ubiquitination and proteolysis of DREB2A, thus allowing the accumulation of sufficient amounts of DREB2A protein to activate downstream gene expression. The mechanisms leading to drought stress-mediated DREB2A accumulation are still unclear. Here, we report that the wheat (Triticum aestivum) TaSAP5 protein, which contains an A20/AN1 domain, acts as an E3 ubiquitin ligase to mediate DRIP degradation and thus increase DREB2A protein levels. Drought induces TaSAP5 expression in wheat, and TaSAP5 overexpression in Arabidopsis and wheat seedlings increased their drought tolerance, as measured by survival rate and grain yield under severe drought stress. TaSAP5 can interact with and ubiquitinate TaDRIP, as well as AtDRIP1 and AtDRIP2, leading to their subsequent degradation through the 26S proteasome pathway. Consistent with this, TaSAP5 overexpression enhances DRIP degradation and increases the levels of DREB2A protein and its downstream targets. These results suggest that TaSAP5 acts to link drought with DREB2A accumulation and illustrate the molecular mechanisms involved in this process.

GCN5 contributes to stem cuticular wax biosynthesis by histone acetylation of CER3 in Arabidopsis.

Cuticular wax is a major component of the surface cuticle of plants, which performs crucial functions in optimizing plant growth. Histone acetylation regulates gene expression in diverse biological processes, but its role in cuticular wax synthesis is not well understood. In this study, we observed that mutations of the Arabidopsis thaliana histone acetyltransferase GENERAL CONTROL NON-REPRESSED PROTEIN5 (GCN5) impaired the accumulation of stem cuticular wax. Three target genes of GCN5, ECERIFERUM3 (CER3), CER26, and CER1-LIKE1 (CER1-L1), were identified by RNA-seq and ChIP assays. H3K9/14 acetylation levels at the promoter regions of CER3, CER26, and CER1-L1 were consistently and significantly decreased in the gcn5-2 mutant as compared to the wild-type. Notably, overexpression of CER3 in the gcn5-2 mutant rescued the defect in stem cuticular wax biosynthesis. Collectively, these data demonstrate that GCN5 is involved in stem cuticular wax accumulation by modulating CER3 expression via H3K9/14 acetylation, which underlines the important role of histone acetylation in cuticular wax biosynthesis.

Proteomic patterns associated with heterosis.

Heterosis is characterized by higher seed yields, plant biomass or other traits in heterozygotes or hybrids compared with their genetically divergent parents, which are often homozygous. Despite extensive investigation of heterosis and its wide application in crops such as maize, rice, wheat and sorghum, its molecular basis is still enigmatic. In the past century, some pioneers have proposed multigene models referring to the complementation of allelic and gene expression variation, which is likely to be an important contributor to heterosis. In addition, there are potential interactions of epigenetic variation involved in heterosis via novel mechanisms. At the level of gene expression, many recent studies have revealed that the heterosis phenomenon can be deciphered not only at the transcriptional level but also at the proteomic level. This review presents an update on the information supporting the involvement of proteomic patterns in heterosis and a possible future direction of the field. This article is part of a Special Issue entitled: Plant Proteomics--a bridge between fundamental processes and crop production, edited by Dr. Hans-Peter Mock.

Histone acetyltransferase general control non-repressed protein 5 (GCN5) affects the fatty acid composition of Arabidopsis thaliana seeds by acetylating fatty acid desaturase3 (FAD3).

Seed oils are important natural resources used in the processing and preparation of food. Histone modifications represent key epigenetic mechanisms that regulate gene expression, plant growth and development. However, histone modification events during fatty acid (FA) biosynthesis are not well understood. Here, we demonstrate that a mutation of the histone acetyltransferase GCN5 can decrease the ratio of α-linolenic acid (ALA) to linoleic acid (LA) in seed oil. Using RNA-Seq and ChIP assays, we identified FAD3, LACS2, LPP3 and PLAIIIß as the targets of GCN5. Notably, the GCN5-dependent H3K9/14 acetylation of FAD3 determined the expression levels of FAD3 in Arabidopsis thaliana seeds, and the ratio of ALA/LA in the gcn5 mutant was rescued to the wild-type levels through the overexpression of FAD3. The results of this study indicated that GCN5 modulated FA biosynthesis by affecting the acetylation levels of FAD3. We provide evidence that histone acetylation is involved in FA biosynthesis in Arabidopsis seeds and might contribute to the optimization of the nutritional structure of edible oils through epigenetic engineering.

Histone acetyltransferase GCN5 is essential for heat stress-responsive gene activation and thermotolerance in Arabidopsis.

Exposure to temperatures exceeding the normal optimum levels, or heat stress (HS), constitutes an environmental disruption for plants, resulting in severe growth and development retardation. Here we show that loss of function of the Arabidopsis histone acetyltransferase GCN5 results in serious defects in terms of thermotolerance, and considerably impairs the transcriptional activation of HS-responsive genes. Notably, expression of several key regulators such as the HS transcription factors HSFA2 and HSFA3, Multiprotein Bridging Factor 1c (MBF1c) and UV-HYPERSENSITIVE 6 (UVH6) is down-regulated in the gcn5 mutant under HS compared with the wild-type. Chromatin immunoprecipitation (ChIP) assays indicated that GCN5 protein is enriched at the promoter regions of HSFA3 and UVH6 genes, but not in HSFA2 and MBF1c, and that GCN5 facilitates H3K9 and H3K14 acetylation, which are associated with HSFA3 and UVH6 activation under HS. Moreover, constitutive expression of UVH6 in the gcn5 mutant partially restores heat tolerance. Taken together, our data indicate that GCN5 plays a key role in the preservation of thermotolerance via versatile regulation in Arabidopsis. In addition, expression of the wheat TaGCN5 gene re-establishes heat tolerance in Arabidopsis gcn5 mutant plants, suggesting that GCN5-mediated thermotolerance may be conserved between Arabidopsis and wheat.

GENERAL CONTROL NONREPRESSED PROTEIN5-Mediated Histone Acetylation of FERRIC REDUCTASE DEFECTIVE3 Contributes to Iron Homeostasis in Arabidopsis.

Iron homeostasis is essential for plant growth and development. Here, we report that a mutation in GENERAL CONTROL NONREPRESSED PROTEIN5 (GCN5) impaired iron translocation from the root to the shoot in Arabidopsis (Arabidopsis thaliana). Illumina high-throughput sequencing revealed 879 GCN5-regulated candidate genes potentially involved in iron homeostasis. Chromatin immunoprecipitation assays indicated that five genes (At3G08040, At2G01530, At2G39380, At2G47160, and At4G05200) are direct targets of GCN5 in iron homeostasis regulation. Notably, GCN5-mediated acetylation of histone 3 lysine 9 and histone 3 lysine 14 of FERRIC REDUCTASE DEFECTIVE3 (FRD3) determined the dynamic expression of FRD3. Consistent with the function of FRD3 as a citrate efflux protein, the iron retention defect in gcn5 was rescued and fertility was partly restored by overexpressing FRD3. Moreover, iron retention in gcn5 roots was significantly reduced by the exogenous application of citrate. Collectively, these data suggest that GCN5 plays a critical role in FRD3-mediated iron homeostasis. Our results provide novel insight into the chromatin-based regulation of iron homeostasis in Arabidopsis.

The TaTCP4/10-B1 cascade regulates awn elongation in wheat (Triticum aestivum L.).

Awns are important morphological markers for wheat and exert a strong physiological effect on wheat yield. The awn elongation suppressor B1 has recently been cloned through association and linkage analysis in wheat. However, the mechanism of awn inhibition centered around B1 remains to be clarified. Here, we identified an allelic variant in the coding region of B1 through analysis of re-sequencing data; this variant causes an amino acid substitution and premature termination, resulting in a long-awn phenotype. Transcriptome analysis indicated that B1 inhibited awn elongation by impeding cytokinin- and auxin-promoted cell division. Moreover, B1 directly repressed the expression of TaRAE2 and TaLks2, whose orthologs have been reported to promote awn development in rice or barley. More importantly, we found that TaTCP4 and TaTCP10 synergistically inhibited the expression of B1, and a G-to-A mutation in the B1 promoter attenuated its inhibition by TaTCP4/10. Taken together, our results reveal novel mechanisms of awn development and provide genetic resources for trait improvement in wheat.

Overexpression of TaLBD16-4D alters plant architecture and heading date in transgenic wheat.

Lateral organ boundaries domain (LBD) proteins, a class of plant-specific transcription factors with a special domain of lateral organ boundaries (LOB), play essential roles in plant growth and development. However, there is little known about the functions of these genes in wheat to date. Our previous study demonstrated that TaLBD16-4D is conducive to increasing lateral root number in wheat. In the present work, we further examined important agronomical traits of the aerial part of transgenic wheat overexpressing TaLBD16-4D. Interestingly, it was revealed that overexpressing TaLBD16-4D could lead to early heading and multiple alterations of plant architecture, including decreased plant height, increased flag leaf size and stem diameter, reduced spike length and tillering number, improved spike density and grain width, and decreased grain length. Moreover, auxin-responsive experiments demonstrated that the expression of TaLBD16-4D in wild-type (WT) wheat plants showed a significant upregulation through 2,4-D treatment. TaLBD16-4D-overexpression lines displayed a hyposensitivity to 2,4-D treatment and reduced shoot gravitropic response. The expressions of a set of auxin-responsive genes were markedly different between WT and transgenic plants. In addition, overexpressing TaLBD16-4D affected the transcript levels of flowering-related genes (TaGI, TaCO1, TaHd1, TaVRN1, TaVRN2, and TaFT1). Notably, the expression of TaGI, TaCO1, TaHd1, TaVRN1, and TaFT1 displayed significant upregulation under IAA treatment. Collectively, our observations indicated that overexpressing TaLBD16-4D could affect aerial architecture and heading time possibly though participating in the auxin pathway.