miR156-PvSPL2 controls culm development by transcriptional repression of switchgrass CYTOKININ OXIDASE/DEHYDROGENASE4.

Culm development in grasses can be controlled by both miR156 and cytokinin. However, the crosstalk between the miR156-SPL module and the cytokinin metabolic pathway remains largely unknown. Here, we found CYTOKININ OXIDASE/DEHYDROGENASE4 (PvCKX4) plays a negative regulatory role in culm development of the bioenergy grass Panicum virgatum (switchgrass). Overexpression of PvCKX4 in switchgrass reduced the internode diameter and length without affecting tiller number. Interestingly, we also found that PvCKX4 was always upregulated in miR156 overexpressing (miR156OE) transgenic switchgrass lines. Additionally, upregulation of either miR156 or PvCKX4 in switchgrass reduced the content of isopentenyl adenine (iP) without affecting trans-zeatin (tZ) accumulation. It is consistent with the evidence that the recombinant PvCKX4 protein exhibited much higher catalytic activity against iP than tZ in vitro. Furthermore, our results showed that miR156-targeted SPL2 bound directly to the promoter of PvCKX4 to repress its expression. Thus, alleviating the SPL2-mediated transcriptional repression of PvCKX4 through miR156 overexpression resulted in a significant increase in cytokinin degradation and impaired culm development in switchgrass. On the contrary, suppressing PvCKX4 in miR156OE transgenic plants restored iP content, internode diameter, and length to wild-type levels. Most strikingly, the double transgenic lines retained the same increased tiller numbers as the miR156OE transgenic line, which yielded more biomass than the wild type. These findings indicate that the miR156-SPL module can control culm development through transcriptional repression of PvCKX4 in switchgrass, which provides a promising target for precise design of shoot architecture to yield more biomass from grasses.

A blueberry MIR156a-SPL12 module coordinates the accumulation of chlorophylls and anthocyanins during fruit ripening.

Color change is an important event during fruit maturation in blueberry, usually depending on chlorophyll degradation and anthocyanin accumulation. MicroRNA156 (miR156)-SPL modules are an important group of regulatory hubs involved in the regulation of anthocyanin biosynthesis. However, little is known regarding their roles in blueberry or in chlorophyll metabolism during color change. In this study, a MIR156 gene (VcMIR156a) was experimentally identified in blueberry (Vaccinium corymbosum). Overexpression of VcMIR156a in tomato (Solanum lycopersicum) enhanced anthocyanin biosynthesis and chlorophyll degradation in the stem by altering pigment-associated gene expression. Further investigation indicated that the VcSPL12 transcript could be targeted by miR156, and showed the reverse accumulation patterns during blueberry fruit development and maturation. Noticeably, VcSPL12 was highly expressed at green fruit stages, while VcMIR156a transcripts mainly accumulated at the white fruit stage when expression of VcSPL12 was dramatically decreased, implying that VcMIR156a-VcSPL12 is a key regulatory hub during fruit coloration. Moreover, VcSPL12 decreased the expression of several anthocyanin biosynthetic and regulatory genes, and a yeast two-hybrid assay indicated that VcSPL12 interacted with VcMYBPA1. Intriguingly, expression of VcSPL12 significantly enhanced chlorophyll accumulation and altered the expression of several chlorophyll-associated genes. Additionally, the chloroplast ultrastructure was altered by the expression of VcMIR156a and VcSPL12. These findings provide a novel insight into the functional roles of miR156-SPLs in plants, especially in blueberry fruit coloration.

The roles of miR156 in abiotic and biotic stresses in plants.

MicroRNAs (miRNAs), known as a kind of non-coding RNA, can negatively regulate its target genes. To date, the roles of various miRNAs in plant development and resistance to abiotic and biotic stresses have been widely explored. The present review summarized and discussed the functions of miR156 or miR156-SPL module in abiotic and biotic stresses, such as drought, salt, heat, cold stress, UV-B radiation, heavy mental hazards, nutritional starvation, as well as plant viruses, plant diseases, etc. Based on this, the regulation of miR156-involved stress tolerance was better understood, thus, it would be much easier for plant biologists to carry out suitable strategies to help plants suffer from unfavorable living environments.

Integrated transcriptome and microRNA sequencing analyses reveal gene responses in poplar leaves infected by the novel pathogen bean common mosaic virus (BCMV).

Recently, a novel poplar mosaic disease caused by bean common mosaic virus (BCMV) was investigated in Populus alba var. pyramidalis in China. Symptom characteristics, physiological performance of the host, histopathology, genome sequences and vectors, and gene regulation at the transcriptional and posttranscriptional levels were analyzed and RT-qPCR (quantitative reverse transcription PCR) validation of expression was performed in our experiments. In this work, the mechanisms by which the BCMV pathogen impacts physiological performance and the molecular mechanisms of the poplar response to viral infection were reported. The results showed that BCMV infection decreased the chlorophyll content, inhibited the net photosynthesis rate (Pn) and stomatal conductance (Gs), and significantly changed chlorophyll fluorescence parameters in diseased leaves. Transcriptome analysis revealed that the expression of the majority of DEGs (differentially expressed genes) involved in the flavonoid biosynthesis pathway was promoted, but the expression of all or almost all DEGs associated with photosynthesis-antenna proteins and the photosynthesis pathway was inhibited in poplar leaves, suggesting that BCMV infection increased the accumulation of flavonoids but decreased photosynthesis in hosts. Gene set enrichment analysis (GSEA) illustrated that viral infection promoted the expression of genes involved in the defense response or plant-pathogen interaction. MicroRNA-seq analysis illustrated that 10 miRNA families were upregulated while 6 families were downregulated in diseased poplar leaves; moreover, miR156, the largest family with the most miRNA members and target genes, was only differentially upregulated in long-period disease (LD) poplar leaves. Integrated transcriptome and miRNA-seq analyses revealed 29 and 145 candidate miRNA-target gene pairs; however, only 17 and 76 pairs, accounting for 2.2% and 3.2% of all DEGs, were authentically negatively regulated in short-period disease (SD) and LD leaves, respectively. Interestingly, 4 miR156/SPL (squamosa promoter-binding-like protein) miRNA-target gene pairs were identified in LD leaves: the miR156 molecules were upregulated, but SPL genes were downregulated. In conclusion, BCMV infection significantly changed transcriptional and posttranscriptional gene expression in poplar leaves, inhibited photosynthesis, increased the accumulation of flavonoids, induced systematic mosaic symptoms, and decreased physiological performance in diseased poplar leaves. This study elucidated the fine-tuned regulation of poplar gene expression by BCMV; moreover, the results also suggested that miR156/SPL modules played important roles in the virus response and development of viral systematic symptoms in plant virus disease.

Wall ingrowth deposition in phloem parenchyma transfer cells in Arabidopsis: Heteroblastic variations and a potential role in pathogen defence.

Transfer cell (TCs) develop unique wall ingrowth networks which amplify plasma membrane surface area and thus maximize nutrient transporter density at key anatomic sites for nutrient exchange within plants and their external environment. These sites fall into 4 main groups corresponding to 4 categories of trans-membrane flux: absorption/secretion of solutes from or to the external environment, and absorption/secretion of solutes from or to internal, extra-cytoplasmic compartments. Research on TC biology over recent decades has demonstrated correlations between wall ingrowth deposition in TCs and enhanced transport capacity in many major agricultural species such as pea, fava bean, cotton and maize. Consequently, there is general consensus that the existence of wall ingrowth morphology implies an augmentation in membrane transport capacity. However, this may not be entirely applicable for phloem parenchyma (PP) TCs in Arabidopsis. Our recent survey of PP TC abundance and distribution in Arabidopsis veins indicated that PP TC development reflects heteroblastic status. A consequence of this observation is the suggestion that PP TCs, or at least wall ingrowth deposition in these cells, potentially act as a physical barrier to defend access of invading pathogens to sugar-rich sieve elements rather than solely in facilitating the export of photoassimilate from collection phloem in leaves.