MicroRNA319-TCP19-IAA3.2 Module Mediates Lateral Root Growth in <i>Populus tomentosa</i>

MicroRNA319 (miR319) and its targets TEOSINTE-BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors are well-characterized regulators of leaf and flower development, yet their role in root development remains elusive. Here, we demonstrated that overexpression of <i>miR319a</i> led to a decrease in the number and density of lateral roots in poplar, while repressing <i>miR319a</i> by short tandem target mimics (STTM) promoted lateral root (LR) development. The auxin signaling repressors <i>IAA3.1</i> and <i>IAA3.2</i> were upregulated in <i>miR319a</i>-OE plants but downregulated in <i>miR319a</i>-STTM plants. After exogenous applications of naphthaleneacetic acid (NAA), which exhibited the characteristics and physiological functions of the endogenous auxin indole-3-acetic acid, the number and density of LR in WT increased by 30% and 44%, respectively. In <i>miR319a</i>-OE plants, the LR number increased by 23% and 48%, and the LR density increased by 10% and 26%. NAA treatment can partially compensate for the phenotype of inhibited LR development caused by the overexpression of <i>miR319a</i>. After N-1-naphthylphthalamic acid (NPA) treatment, which is a key inhibitor of the directional (polar) transport of the auxin hormone in plants, the LR number in WT decreased by 70%. In the overexpression plants, the number of lateral roots decreased by 85–87%, and in the STTM plants, the number of lateral roots decreased by about 83%. It was proved that NPA treatment could reverse the phenotype of increased LR number in <i>miR319a</i>-STTM plants. Expression analysis revealed that miR319a significantly inhibited the expression of the key auxin-regulated genes <i>IAA3.1</i> and <i>IAA3.2</i>, suggesting that auxin signaling might mediate its effects on lateral root formation. Additionally, we compared the fluorescence signal in the reporter line with GFP expression driven by the auxin-responsive DR5 promoter within the genetic backgrounds of WT, <i>miR319a</i>-OE, and <i>miR319a</i>-STTM plants, which revealed that auxin signaling was stronger in the epidermal cells and elongation zone cells in the LR of <i>miR319a</i>-OE plants, whereas in LR of WT and <i>miR319a</i>-STTM plants, auxin signaling was more pronounced in the root tip meristematic cells. Furthermore, transactivation assays and expression analysis indicated that <i>IAA3.2</i> was a downstream target of TCP19. Chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) confirmed that TCP19 directly bound to the promoter region of <i>IAA3.2</i>. These findings establish that miR319a targeted and cleaved <i>TCP19</i>, and TCP19 further directly and negatively regulates the expression of <i>IAA3.2</i>, thereby controlling LR development in <i>Populus tomentosa</i> (<i>P. tomentosa</i>). The formation of LR can expand the plant root system, which is of great significance for the vegetative propagation of plants and the in-vitro regeneration of explants. Moreover, the formation of LR is an important strategy for plants to cope with environmental stresses. This study provides a theoretical basis for breeding poplars more suitable for vegetative propagation.