Coordination of miR319-TaPCF8 with TaSPL14 orchestrates auxin signaling and biosynthesis to regulate plant height in common wheat.

Wheat culms, comprising four to six internodes, are critically involved in determining plant height and lodging resistance, essential factors for field performance and regional adaptability. This study revealed the regulatory function of miR319 in common wheat plant height. Repression of tae-miR319 through short tandem target mimics (STTM) caused an increased plant height, while overexpression (OE) of tae-miR319 had the opposite effect. Overexpressing a miR319-resistant target gene TaPCF8 (rTaPCF8), increased plant height. TaPCF8 acted as a transcription repressor of downstream genes TaIAAs, which interact physically with TaSPL14. The significant differences of indole-3-acetic acid (IAA) contents indicate the involvement of auxin pathway in miR319-mediated plant height regulation. Finally, we identified two TaPCF8 haplotypes in global wheat collections. TaPCF8-5A-Hap2, as per association and evolution examinations, was subjected to strong substantial selection throughout wheat breeding. This haplotype, associated with shorter plant height, aligns with global breeding requirements. Consequently, in high-yield wheat breeding, we proposed a potential molecular marker for marker-assisted selection (MAS). Our findings offer fresh perspectives into the molecular mechanisms that underlie the miR319-TaPCF8 module's regulation of plant height by orchestrating auxin signaling and biosynthesis in wheat.

Strigolactones promote flowering by inducing the miR319-LA-SFT module in tomato.

Strigolactones are a class of phytohormones with various functions in plant development, stress responses, and in the interaction with (micro)organisms in the rhizosphere. While their effects on vegetative development are well studied, little is known about their role in reproduction. We investigated the effects of genetic and chemical modification of strigolactone levels on the timing and intensity of flowering in tomato (Solanum lycopersicum L.) and the molecular mechanisms underlying such effects. Results showed that strigolactone levels in the shoot, whether endogenous or exogenous, correlate inversely with the time of anthesis and directly with the number of flowers and the transcript levels of the florigen-encoding gene SINGLE FLOWER TRUSS (SFT) in the leaves. Transcript quantifications coupled with metabolite analyses demonstrated that strigolactones promote flowering in tomato by inducing the activation of the microRNA319-LANCEOLATE module in leaves. This, in turn, decreases gibberellin content and increases the transcription of SFT. Several other floral markers and morpho-anatomical features of developmental progression are induced in the apical meristems upon treatment with strigolactones, affecting floral transition and, more markedly, flower development. Thus, strigolactones promote meristem maturation and flower development via the induction of SFT both before and after floral transition, and their effects are blocked in plants expressing a miR319-resistant version of LANCEOLATE. Our study positions strigolactones in the context of the flowering regulation network in a model crop species.

Blocking miR396 activity by overexpression MIM396 improved switchgrass tiller number and biomass yield.

BACKGROUND: MicroRNA396 (miR396) plays an important role in the regulation of plant growth and development by repressing the expression level of its target growth-regulating factor (GRF) family genes. In our previous study, we found that overexpression of miR396 negatively regulated both tillering and biomass yield in switchgrass (Panicum virgatum L.). We, therefore, speculated that blocking the expression of miR396 could enhance switchgrass tillering and biomass yield. Here, we produced transgenic switchgrass plants overexpressing a target mimicry form of miR396 (MIM396) in wild type (WT) and Os-MIR319b overexpressing switchgrass plant (with higher enzymatic hydrolysis efficiency, but reduced tillering), in which the expression of miR396 was blocked. The phenotype and biological yields of these plants were analyzed. RESULTS: Blocking miR396 to improve its target PvGRFs expression in switchgrass improved the tiller number and dry weight of transgenic plants. Further morphological analysis revealed that MIM396 plants increased the number of aerial branches and basal tillers compared to those of wild-type plants. The enzymatic efficiency of MIM396 plants was reduced; however, the total sugar production per plant was still significantly higher than that of wild-type plants due to the increase in biomass. In addition, blocking miR396 in a transgenic switchgrass plant overexpressing Os-MIR319b (TG21-Ms) significantly increased the PvGRF1/3/5 expression level and tiller number and biomass yield. The miR156-target gene PvSPL4, playing a negative role in aerial and basal buds outgrowth, showed significant downregulated in MIM396 and TG21-Ms. Those results indicate that miR396-PvGRFs, through disrupting the PvSPL4 expression, are involved in miR319-PvPCFs in regulating tiller number, at least partly. CONCLUSIONS: MIM396 could be used as a molecular tool to improving tiller number and biomass yield in switchgrass wild type and miR319b transgenic plants. This finding may be applied to other graminaceous plants to regulate plant biological yield.

The cotton MYB33 gene is a hub gene regulating the trade-off between plant growth and defense in Verticillium dahliae infection.

INTRODUCTION: Sessile plants engage in trade-offs between growth and defense capacity in response to fluctuating environmental cues. MYB is an important transcription factor that plays many important roles in controlling plant growth and defense. However, the mechanism behind how it keeps a balance between these two physiological processes is still largely unknown. OBJECTIVES: Our work focuses on the dissection of the molecular mechanism by which GhMYB33 regulates plant growth and defense. METHODS: The CRISPR/Cas9 technique was used to generate mutants for deciphering GhMYB33 functions. Yeast two-hybrid, luciferase complementary imaging, and co-immunoprecipitation assays were used to prove that proteins interact with each other. We used the electrophoretic mobility shift assay, yeast one-hybrid, and luciferase activity assays to analyze GhMYB33 acting as a promoter. A ß-glucuronidase fusion reporter and 5' RNA ligase mediated amplification of cDNA ends analysis showed that ghr-miR319c directedly cleaved the GhMYB33 mRNA. RESULTS: Overexpressing miR319c-resistant GhMYB33 (rGhMYB33) promoted plant growth, accompanied by a significant decline in resistance against Verticillium dahliae. Conversely, its knockout mutant, ghmyb33, demonstrated growth restriction and concomitant augmentation of V. dahliae resistance. GhMYB33 was found to couple with the DELLA protein GhGAI1 and bind to the specific cis-elements of GhSPL9 and GhDFR1 promoters, thereby modulating internode elongation and plant resistance in V. dahliae infection. The ghr-miR319c was discovered to target and suppress GhMYB33 expression. The overexpression of ghr-miR319c led to enhanced plant resistance and a simultaneous reduction in plant height. CONCLUSION: Our findings demonstrate that GhMYB33 encodes a hub protein and controls the expression of GhSPL9 and GhDFR1, implicating a pivotal role for the miR319c-MYB33 module to regulate the trade-offs between plant growth and defense.

Regulation of miR319b-Targeted SlTCP10 during the Tomato Response to Low-Potassium Stress.

Potassium deficiency confines root growth and decreases root-to-shoot ratio, thereby limiting root K+ acquisition. This study aimed to identify the regulation network of microRNA319 involved in low-K+ stress tolerance in tomato (Solanum lycopersicum). SlmiR319b-OE roots demonstrated a smaller root system, a lower number of root hairs and lower K+ content under low-K+ stress. We identified SlTCP10 as the target of miR319b using a modified RLM-RACE procedure from some SlTCPs' predictive complementarity to miR319b. Then, SlTCP10-regulated SlJA2 (an NAC transcription factor) influenced the response to low-K+ stress. CR-SlJA2 (CRISPR-Cas9-SlJA2) lines showed the same root phenotype to SlmiR319-OE compared with WT lines. OE-SlJA2(Overexpression-SlJA2) lines showed higher root biomass, root hair number and K+ concentration in the roots under low-K+ conditions. Furthermore, SlJA2 has been reported to promote abscisic acid (ABA) biosynthesis. Therefore, SlJA2 increases low-K+ tolerance via ABA. In conclusion, enlarging root growth and K+ absorption by the expression of SlmiR319b-regulated SlTCP10, mediating SlJA2 in roots, could provide a new regulation mechanism for increasing K+ acquisition efficiency under low-K+ stress.

miR319 and its target TCP4 involved in plant architecture regulation in Brassica napus.

Plant architecture is a collection of genetically controlled crop productivity and adaptation. MicroRNAs (miRNAs) have been proved to function in various biological processes, but little is known about how miRNA regulates plant architecture in rapeseed (Brassica napus L.). In this study, four small RNA libraries and two degradome libraries from shoot apex of normal and rod-like plants were sequenced. A total of 639 miRNA precursors and 16 differentially expressed miRNAs were identified in this study. In addition, 322 targets were identified through degradome sequencing. Among them, 14 targets were further validated via RNA ligase-mediated 5' rapid amplification of cDNA ends. Transgenic approach showed that increased TCP4 activity in Arabidopsis resulted in premature onset of maturation and reduced plant size along with early flowering and shortened flowering time. miR319-OE lines in Brassica napus exhibited serrated leaves and abnormal development of shoot apical meristem (SAM), which led to the deformed growth of stem and reduced plant height. In conclusion, our study lays the foundation for elucidating miRNA regulate plant architecture and provides new insight into the miR319/TCP4 module regulates plant architecture in rapeseed.

miR319-Regulated TCP3 Modulates Silique Development Associated with Seed Shattering in Brassicaceae.

Seed shattering is an undesirable trait that leads to crop yield loss. Improving silique resistance to shattering is critical for grain and oil crops. In this study, we found that miR319-targeted TEOSINTE BRANCHED 1, CYCLOIDEA, and PROLIFERATING CELL NUCLEAR ANTIGEN BINDING FACTOR (TCPs) inhibited the process of post-fertilized fruits (silique) elongation and dehiscence via regulation of FRUITFULL (FUL) expression in Arabidopsis thaliana and Brassica napus. AtMIR319a activation resulted in a longer silique with thickened and lignified replum, whereas overexpression of an miR319a-resistant version of AtTCP3 (mTCP3) led to a short silique with narrow and less lignified replum. Further genetic and expressional analysis suggested that FUL acted downstream of TCP3 to negatively regulate silique development. Moreover, hyper-activation of BnTCP3.A8, a B. napus homolog of AtTCP3, in rapeseed resulted in an enhanced silique resistance to shattering due to attenuated replum development. Taken together, our findings advance our knowledge of TCP-regulated silique development and provide a potential target for genetic manipulation to reduce silique shattering in Brassica crops.

Cotton miR319b-Targeted TCP4-Like Enhances Plant Defense Against Verticillium dahliae by Activating GhICS1 Transcription Expression.

Teosinte branched1/Cincinnata/proliferating cell factor (TCP) transcription factors play important roles in plant growth and defense. However, the molecular mechanisms of TCPs participating in plant defense remain unclear. Here, we characterized a cotton TCP4-like fine-tuned by miR319b, which could interact with NON-EXPRESSER OF PATHOGEN-RELATED GENES 1 (NPR1) to directly activate isochorismate synthase 1 (ICS1) expression, facilitating plant resistance against Verticillium dahliae. mRNA degradome data and GUS-fused assay showed that GhTCP4-like mRNA was directedly cleaved by ghr-miR319b. Knockdown of ghr-miR319b increased plant resistance to V. dahliae, whereas silencing GhTCP4-like increased plant susceptibility by the virus-induced gene silencing (VIGS) method, suggesting that GhTCP4-like is a positive regulator of plant defense. According to the electrophoretic mobility shift assay and GUS reporter analysis, GhTCP4-like could transcriptionally activate GhICS1 expression, resulting in increased salicylic acid (SA) accumulation. Yeast two-hybrid and luciferase complementation image analyses demonstrated that GhTCP4-like interacts with GhNPR1, which can promote GhTCP4-like transcriptional activation in GhICS1 expression according to the GUS reporter assay. Together, these results revealed that GhTCP4-like interacts with GhNPR1 to promote GhICS1 expression through fine-tuning of ghr-miR319b, leading to SA accumulation, which is percepted by NPR1 to increase plant defense against V. dahliae. Therefore, GhTCP4-like participates in a positive feedback regulation loop of SA biosynthesis via NPR1, increasing plant defenses against fungal infection.

The miR319/TaGAMYB3 module regulates plant architecture and improves grain yield in common wheat (Triticum aestivum).

Plant architecture is a key determinant of crop productivity and adaptation. The highly conserved microRNA319 (miR319) family functions in various biological processes, but little is known about how miR319 regulates plant architecture in wheat (Triticum aestivum). Here, we determined that the miR319/TaGAMYB3 module controls plant architecture and grain yield in common wheat. Repressing tae-miR319 using short tandem target mimics resulted in favorable plant architecture traits, including increased plant height, reduced tiller number, enlarged spikes and flag leaves, and thicker culms, as well as enhanced grain yield in field plot tests. Overexpressing tae-miR319 had the opposite effects on plant architecture and grain yield. Although both TaPCF8 and TaGAMYB3 were identified as miR319 target genes, genetic complementation assays demonstrated that only miR319-resistant TaGAMYB3 (rTaGAMYB3) abolished tae-miR319-mediated growth inhibition of flag leaves and spikes. TaGAMYB3 functions as a transcriptional activator of downstream genes, including TaPSKR1, TaXTH23, TaMADS5 and TaMADS51, by binding to their promoters. Furthermore, TaGAMYB3 physically interacts with TaBA1, an important regulator of spike development, to additively activate the transcription of downstream genes such as TaMADS5. Our findings provide insight into how the miR319/TaGAMYB3 module regulates plant architecture and improves grain yield in common wheat.

miR319a targeting of CsTCP10 plays an important role in defense against gray blight disease in tea plant (Camellia sinensis).

Gray blight disease occurs widely in major tea-producing areas and harms the leaves of tea trees, which affects the quality and yield of processed tea. According to an analysis of previous sequencing data, miR319a may be important in the resistance of tea plants to gray blight disease. In this study, based on 5'RLM-RACE, qRT-PCR, sODN, CIN and transient transformation experiments in tobacco, CsTCP10 and CsTCP4 were found to be cleaved by miR319a. qRT-PCR and northern blotting also revealed that the expression pattern of CsTCP10 in tea leaves was opposite to that of miR319a, while that of CsTCP4 displayed no similar change. Furthermore, a large amount of reactive oxygen species was found to accumulate in tea leaves in the antisense oligodeoxynucleotide experiment, while the expression of CsTCP10 was inhibited. These results suggest that CsTCP10 is a positive regulator of the resistance of tea plants to gray blight disease. Compared with the wild-type, the expression of AtTCP10 in transgenic Arabidopsis plants was downregulated. After infection with the pathogen, the transgenic plants were more severely damaged. Our results suggest that miR319a facilitates Pestalotiopsis infection by suppressing the expression of CsTCP10 in tea plants.

Potent inhibition of TCP transcription factors by miR319 ensures proper root growth in Arabidopsis.

KEY MESSAGE: Proper root growth depends on the clearance of TCP transcripts from the root apical meristem by microRNA miR319. The evolutionarily conserved microRNA miR319 regulates genes encoding TCP transcription factors in angiosperms. The miR319-TCP module controls cell proliferation and differentiation in leaves and other aerial organs. The current model sustains that miR319 quantitatively tunes TCP activity during leaf growth and development, ultimately affecting its size. In this work we studied how this module participates in Arabidopsis root development. We found that misregulation of TCP activity through impairment of miR319 binding decreased root meristem size and root length. Cellular and molecular analyses revealed that high TCP activity affects cell number and cyclin expression but not mature cell length, indicating that, in roots, unchecking the expression of miR319-regulated TCPs significantly affects cell proliferation. Conversely, tcp multiple mutants showed no obvious effect on root growth, but strong defects in leaf morphogenesis. Therefore, in contrast to the quantitative regulation of the TCPs by miR319 in leaves, our data suggest that miR319 clears TCP transcripts from root cells. Hence, we provide new insights into the functions of the miR319-TCP regulatory system in Arabidopsis development, highlighting a different modus operandi for its action mechanism in roots and shoots.

Overexpression of Mtr-miR319a Contributes to Leaf Curl and Salt Stress Adaptation in Arabidopsis thaliana and Medicago truncatula.

Salt stress is a worldwide agronomic issue that limits crop yield and quality. Improving salt stress tolerance via genetic modification is the most efficient method to conquer soil salinization problems in crops. Crop miRNAs have been declared to be tightly associated with responding and adapting to salt stress and are advantageous for salt tolerance modification. However, very few studies have validated vital salt tolerance miRNAs and coupled potent target genes in Medicago species, the most economically important forage legume species. In this study, Mtr-miR319a, a miRNA that was identified from the previous next-generation sequencing assay of salt-treated Medicago truncatula, was overexpressed in M. truncatula and Arabidopsis thaliana, inducing the curly leaves and salt stress tolerance phenotypes. Combining the elevated expression level of Mtr-miR319a in the M. truncatula overexpression lines under normal and salt-treatment conditions, the regulatory roles of Mtr-miR319a in leaf development and salt stress adaptation were demonstrated. Several predicted target genes of Mtr-miR319a were also regulated by Mtr-miR319a and were associated with the aforementioned phenotypes in M. truncatula plants, most notably MtTCP4. Our study clarified the functional role of Mtr-miR319a and its target genes in regulating leaf development and defending salt stress, which can help to inform crop breeding efforts for improving salt tolerance via genetic engineering.

Genome-Wide Identification of TCP Transcription Factors Family in Sweet Potato Reveals Significant Roles of miR319-Targeted TCPs in Leaf Anatomical Morphology.

Plant-specific TCP transcription factors play vital roles in the controlling of growth, development, and the stress response processes. Extensive researches have been carried out in numerous species, however, there hasn't been any information available about TCP genes in sweet potato (Ipomoea batatas L.). In this study, a genome-wide analysis of TCP genes was carried out to explore the evolution and function in sweet potato. Altogether, 18 IbTCPs were identified and cloned. The expression profiles of the IbTCPs differed dramatically in different organs or different stages of leaf development. Furthermore, four CIN-clade IbTCP genes contained miR319-binding sites. Blocking IbmiR319 significantly increased the expression level of IbTCP11/17 and resulted in a decreased photosynthetic rate due to the change in leaf submicroscopic structure, indicating the significance of IbmiR319-targeted IbTCPs in leaf anatomical morphology. A systematic analyzation on the characterization of the IbTCPs together with the primary functions in leaf anatomical morphology were conducted to afford a basis for further study of the IbmiR319/IbTCP module in association with leaf anatomical morphology in sweet potato.

Sequence and functional analysis of MIR319 promoter homologs from Brassica juncea reveals regulatory diversification and altered expression under stress.

KEY MESSAGE: Extensive regulatory divergence during development, abiotic stress and ABA regime observed amongst promoter homologs and homeologs of MIR319 from Brassica juncea. Gene duplication followed by sub-functionalization, neo-functionalization, and pseudogenization are routes to functional and adaptive diversification. The influence of polyploidy on protein-coding genes is well investigated but little is known about their impact on transcriptional regulation of MIRNA gene family. The present study was therefore performed with an aim to uncover regulatory diversification of MIR319 homologs and homeologs in Brassica juncea. We employed comparative genomics to identify and isolate six promoter homologs of MIR319 from B. juncea. Regulatory diversification was studied using analysis of reporter activity driven by BjMIR319 promoters in a heterologous system employing promoter-reporter fusion constructs. MIR319 is known to play important roles in leaf and flower development, and multiple stress responses. Reporter activity was therefore monitored during development, hormonal and stress regimes. In-silico analyses revealed differential distribution of cis-regulatory motifs and functional analysis revealed distinct spatiotemporal expression patterns. The significance of presence of selected cis-regulatory motifs corresponding to heat, cold, salt and ABA stress were further functionally validated. It was observed that promoter of Bj -MIR319a-A01 was upregulated in response to cold and salt stress, while promoter of Bj -MIR319c-A04 (D1) and Bj -MIR319c-A05 (FL) were downregulated in response to high temperature. In summary, comparative analysis of homologous promoters from Brassica juncea, an allopolyploid revealed extensive sequence and functional diversity. Spatiotemporal activity of reporter gene driven by BjMIR319 promoter was distinct, and partially overlapping with from those reported previously for A. thaliana. The present study clearly demonstrates regulatory divergence amongst promoter homologs of MIR319 in Brassica juncea during development and stress response, and underlines the urgent need for dissection of promoter function and detailed characterization including identification of interacting trans-factors. Genbank accession numbers: MT379853-MT379858.

MicroRNA319a regulates plant resistance to Sclerotinia stem rot.

MicroRNA319a (miR319a) controls cell division arrest in plant leaves by inhibiting the expression of TCP (TEOSINTE BRANCHED 1/CYCLOIDEA/PCF) family genes. However, it is unclear whether miR319a influences infection by necrotrophic pathogens and host susceptibility. In this study, we revealed that miR319a affects plant resistance to stem rot disease caused by Sclerotinia sclerotiorum. In Brassica rapa plants infected with S. sclerotiorum, miR319a levels increased while the expression levels of several BraTCP genes significantly decreased compared with those of uninfected plants. Overexpression of BraMIR319a in B. rapa increased the susceptibility of the plants to S. sclerotiorum and aggravated stem rot disease, whereas overexpression of BraTCP4-1 promoted plant resistance. RNA sequencing data revealed a potential relationship between miR319a and pathogen-related WRKY genes. Chromatin immunoprecipitation, electrophoretic mobility shift, and reporter transaction assays showed that BraTCP4-1 could bind to the promoters of WRKY75, WRKY70, and WRKY33 and directly activate these pathogen-related genes. Moreover, the expression levels of WRKY75, WRKY70, and WRKY33 in plants overexpressing BraMIR319a decreased significantly, whereas those of plants overexpressing BraTCP4-1 increased significantly, relative to the wild type. These results suggest that miR319a and its target gene BraTCP4 control stem rot resistance through pathways of WRKY genes.

miR319c acts as a positive regulator of tomato against Botrytis cinerea infection by targeting TCP29.

miR319 family is one of the oldest and most conservative miRNA families in plant and plays an important role in plant development and abiotic stress response. In our previous study, the abundance of sly-miR319c was increased in tomatoes infected by B. cinerea, but the roles and regulatory mechanisms of sly-miR319c in B. cinerea-infected tomato remain unclear. In this study, we confirmed that miR319c was increased in tomato with B. cinerea infection. In contrast, A TCP transcript factor, TCP29, targeted by sly-miR319c was decreased in B. cinerea-infected tomato. Therefore, transgenic Arabidopsis overexpressing sly-miR319c or its target were generated for understanding the biological roles and molecular mechanism of miR319c in B.cinerea-infected plants. Results showed that miR319c overexpression improved the resistance of transgenic plants to B. cinerea, whereas TCP29 overexpression increased the susceptibility of transgenic plant to B. cinerea. So far, TCP transcription factors have been reported mainly in developmental processes. Our data indicate that TCP29 act as a negative regulator to B.cinerea infection. In conclusion, our results indicate that sly-miR319c is a positive regulator of tomato resistance to B. cinerea infection by targeting TCP29.

MiR319a-targeted PtoTCP20 regulates secondary growth via interactions with PtoWOX4 and PtoWND6 in Populus tomentosa.

Secondary growth is a key characteristic of trees, which requires the coordination of multiple regulatory mechanisms including transcriptional regulators and microRNAs (miRNAs). However, the roles of microRNAs in the regulation of secondary growth need to be explored in depth. Here, the role of miR319a and its target, PtoTCP20, in the secondary growth of Populus tomentosa stem was investigated using genetic and molecular analyses. The expression level of miR319a gradually decreased from primary to secondary growth in P. tomentosa, while that of PtoTCP20 gradually increased. MiR319a overexpression in seedlings resulted in delayed secondary growth and decreased xylem production, while miR319a knockdown and PtoTCP20 overexpression promoted secondary growth and increased xylem production. Further analysis showed that PtoTCP20 interacted with PtoWOX4a and activated PtoWND6 transcription in vitro and in vivo. Our data show that PtoTCP20 controls vascular cambium proliferation by binding to PtoWOX4a, and promotes secondary xylem differentiation by activating PtoWND6 transcription, thereby regulating secondary growth in P. tomentosa. Our findings provide insight into the molecular mechanisms underlying secondary growth in trees.

De Novo Shoot Regeneration Controlled by HEN1 and TCP3/4 in Arabidopsis.

Plants have the ability to regenerate whole plant body parts, including shoots and roots, in vitro from callus derived from a variety of tissues. However, the underlying mechanisms for this de novo organogenesis, which is based on the totipotency of callus cells, are poorly understood. Here, we report that a microRNA (miRNA)-mediated posttranscriptional regulation plays an important role in de novo shoot regeneration. We found that mutations in HUA ENHANCER 1 (HEN1), a gene encoding a small RNA methyltransferase, cause cytokinin-related defects in de novo shoot regeneration. A hen1 mutation caused a large reduction in the miRNA319 (miR319) level and a subsequent increase in its known target (TCP3 and TCP4) transcript levels. TCP transcription factors redundantly inhibited shoot regeneration and directly activated the expression of a negative regulator of cytokinin response ARABIDOPSIS THALIANA RESPONSE REGULATOR 16 (ARR16). A tcp4 mutation at least partly rescued the shoot-regeneration defect and derepression of ARR16 in hen1. These findings demonstrate that the miR319-TCP3/4-ARR16 axis controls de novo shoot regeneration by modulating cytokinin responses.

Genetic interactions among Pto-miR319 family members and their targets influence growth and wood properties in Populus tomentosa.

MicroRNAs (miRNAs) play crucial roles in all aspects of plant growth and development, but the genetic interactions of miRNAs and their target genes in woody plants are largely unknown. Here, we integrated association genetics and expression profiling to decipher the allelic variations and interactions of the Pto-MIR319 family of miRNAs and 12 putative Pto-miR319 target genes related to wood formation in 435 unrelated individuals of Populus tomentosa Carrière (Chinese white poplar). Expression pattern analysis showed that among all pairings between expressions of pre-miRNA of Pto-MIR319 members and targets, 70.0% showed negative correlation of expression levels (r = - 0.944 to 0.674, P < 0.01) in eight tissues and organs of poplar, suggesting that Pto-miR319 may participate in the regulatory network of wood formation. Single SNP-based association studies identified 137 significant associations (P < 0.01, Q < 0.1), representing 126 unique SNPs from Pto-MIR319 members and their targets, with 10 tree growth traits, revealing that these genetic factors have common roles related to wood formation. Epistasis analysis uncovered 105 significant SNP-SNP associations (P < 0.01) influencing the 10 traits, demonstrating the close genetic interactions between Pto-MIR319 family members and the 12 Pto-miR319 target genes. Notably, one common SNP, in the precursor region of Pto-MIR319e, affected the stability of Pto-MIR319e's secondary structure by altering the stem-loop structure and minimum free energy, contributing to variations in the expression of Pto-MIR319e and Pto-miR319e target genes. This study enriches the understanding of the functions of miR319 family miRNAs in poplar and exemplifies a feasible approach to exploring the genetic effects underlying miRNA-mRNA interactions related to complex traits in trees.

miR319a/TCP module and DELLA protein regulate trichome initiation synergistically and improve insect defenses in Populus tomentosa.

Trichomes are specialized epidermal cells that contribute to plant resistance against herbivores. Their formation is controlled precisely by multiple genetic and environmental signals. Previous studies have shown that microRNA319 (miR319) and gibberellin (GA) signaling are involved in trichome development in Arabidopsis, but little is known about their interaction between these factors. Here we reported that the miR319a/TEOSINTE BRANCHED/CYCLOIDEA/PCF (TCP) module participates in trichome initiation synergistically with GA signaling in Populus tomentosa. We demonstrated that overexpression of miR319a decreased transcription levels of its targeted TCPs and significantly elevated leaf trichome density in transgenic poplar, resulting in decreasing insect herbivory. Conversely, repressing miR319a by short tandem target mimics (STTM) elevated TCP expression levels and decreased trichome density in transgenic plants. The trichome phenotype of 35S:miR319a plants could be abolished by introducing a miR319a-resistant form of TCP19. Furthermore, the miR319a-targeted TCP19 interacted directly with REPRESSOR OF ga1-3 (RGA), a downstream repressor of GA signaling. TCP19 and RGA synergistically inhibited the GLABROUS1 (GL1)-induced expression of trichome marker gene GLABRA2 (GL2), thereby repressing leaf trichome initiation. Our results provide an insight into the molecular mechanism by which miR319/TCP19 module and GA signaling coordinated regulating trichome initiation in P. tomentosa.