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Root growth is modulated by different factors, including phytohormones, transcription factors, and microRNAs (miRNAs). MicroRNA156 and its targets, the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes, define an age-dependent pathway that controls several developmental processes, including lateral root emergence. However, it remains unclear whether miR156-regulated SPLs control root meristem activity and root-derived de novo shoot regeneration. Here, we show that MIR156 and SPL genes have opposing expression patterns during the progression of primary root (PR) growth in Arabidopsis, suggesting that age cues may modulate root development. Plants with high miR156 levels display reduced meristem size, resulting in shorter primary root (PRs). Conversely, plants with reduced miR156 levels show higher meristem activity. Importantly, loss of function of SPL10 decreases meristem activity, while SPL10 de-repression increases it. Meristem activity is regulated by SPL10 probably through the reduction of cytokinin responses, via the modulation of type-B ARABIDOPSIS RESPONSE REGULATOR1(ARR1) expression. We also show that SPL10 de-repression in the PRs abolishes de novo shoot regenerative capacity by attenuating cytokinin responses. Our results reveal a cooperative regulation of root meristem activity and root-derived de novo shoot regeneration by integrating age cues with cytokinin responses via miR156-targeted SPL10.

Summary Salt stress dramatically impedes plant growth and development as well as crop yield. The apple production regions are reduced every year, because of the secondary salt damage by improper fertilization and irrigation. To expand the cultivation area of apple (Malus domestica) and select salt‐resistant varieties, the mechanism of salt tolerance in apple is necessary to be clarified. The miR156/SPL regulatory module plays key roles in embryogenesis, morphogenesis, life cycle stage transformation, flower formation and other processes. However, its roles in the mechanisms of salt tolerance are unknown. In order to elucidate the mechanism of 156/SPL regulating salt stress in apple, we performed RLM‐5’ RACE and stable genetic transformation technology to verify that both mdm‐MIR156a and MdSPL13 responded to salt stress in apple and that the latter was the target of the former. MIR156a overexpression weakened salt resistance in apple whereas MdSPL13 overexpression strengthened it. A total of 6094 differentially expressed genes relative to nontransgenic apple plants were found by RNA‐Seq analysis of MdSPL13OE. Further verification indicated that MdSPL13 targeted the MdWRKY100 gene promoter. Moreover, MdWRKY100 overexpression enhanced salt tolerance in apple. Our results revealed that the miR156/SPL module regulates salt tolerance by up‐regulating MdWRKY100 in apple. This study is the first to elucidate the mechanism underlying the miRNA network response to salt stress in apple and provides theoretical and empirical bases and genetic resources for the molecular breeding of salt tolerance in apple.

Summary Increase in grain yield is always a major objective of wheat genetic improvement. The SQUAMOSA promoter‐binding protein‐like (SPL) genes, coding for a small family of diverse plant‐specific transcription factors, represent important targets for improving grain yield and other major agronomic traits in rice. The function of the SPL genes in wheat remains to be investigated in this respect. In this study, we identified 56 wheat orthologues of rice SPL genes belonging to 19 homoeologous groups. Like in rice, nine orthologous TaSPL genes harbour the microRNA156 recognition elements (MRE) in their last exons except for TaSPL13, which harbour the MRE in its 3′‐untranslated region (3′UTR). We modified the MRE of TaSPL13 using CRISPR‐Cas9 and generated 12 mutations in the three homoeologous genes. As expected, the MRE mutations led to an approximately two‐fold increase in the TaSPL13 mutant transcripts. The phenotypic evaluation showed that the MRE mutations in TaSPL13 resulted in a decrease in flowering time, tiller number, and plant height, and a concomitantly increase in grain size and number. The results show that the TaSPL13 mutants exhibit a combination of different phenotypes observed in Arabidopsis AtSPL3/4/5 mutants and rice OsSPL13/14/16 mutants and hold great potential in improving wheat yield by simultaneously increasing grain size and number and by refining plant architecture. The novel TaSPL13 mutations generated can be utilized in wheat breeding programmes to improve these agronomic traits.

Main conclusionThe hop phenological cycle was described in subtropical condition of Brazil showing that flowering can happen at any time of year and this was related to developmental molecular pathways.Hops are traditionally produced in temperate regions, as it was believed that vernalization was necessary for flowering. Nevertheless, recent studies have revealed the potential for hops to flower in tropical and subtropical climates. In this work, we observed that hops in the subtropical climate of Minas Gerais, Brazil grow and flower multiple times throughout the year, independently of the season, contrasting with what happens in temperate regions. This could be due to the photoperiod consistently being inductive, with daylight hours below the described threshold (16.5 h critical). We observed that when the plants reached 7–9 nodes, the leaves began to transition from heart-shaped to trilobed-shaped, which could be indicative of the juvenile to adult transition. This could be related to the fact that the 5th node (in plants with 10 nodes) had the highest expression of miR156, while two miR172s increased in the 20th node (in plants with 25 nodes). Hop flowers appeared later, in the 25th or 28th nodes, and the expression of HlFT3 and HlFT5 was upregulated in plants between 15 and 20 nodes, while the expression of HlTFL3 was upregulated in plants with 20 nodes. These results indicate the role of axillary meristem age in regulating this process and suggest that the florigenic signal should be maintained until the hop plants bloom. In addition, it is possible that the expression of TFL is not sufficient to inhibit flowering in these conditions and promote branching. These findings suggest that the reproductive transition in hop under inductive photoperiodic conditions could occur in plants between 15 and 20 nodes. Our study sheds light on the intricate molecular mechanisms underlying hop floral development, paving the way for potential advancements in hop production on a global scale.

Drought stress is the main cause of agricultural crop loss in the world. However, plants have developed mechanisms that allow them to tolerate drought stress conditions. At cellular level, drought stress induces changes in metabolite accumulation, including increases in anthocyanin levels due to upregulation of the anthocyanin biosynthetic pathway. Recent studies suggest that the higher anthocyanin content observed under drought stress conditions could be a consequence of a rise in the abscisic acid (ABA) concentration. This plant hormone crosses the plasma membrane by specific transporters, and it is recognized at the cytosolic level by receptors known as pyrabactin resistance (PYR)/regulatory component of ABA receptors (PYR/RCARs) that regulatedownstream components. In this review, we discuss the hypothesis regarding the involvement of ABA in the regulation of microRNA156 (miRNA156), which is upregulated as part of dehydration stress responsiveness in different species. The miRNA156 upregulation produces a greater level of anthocyanin gene expression, forming the multienzyme complex that will synthesize an increased level of anthocyanins at the cytosolic face of the rough endoplasmic reticulum (RER). After synthesis, anthocyanins are transported from the RER to the vacuole by two possible models of transport: (1) membrane vesicle-mediated transport, or (2) membrane transporter-mediated transport. Thus, the aim was to analyze the recent findings on synthesis, transport and the possible mechanism by which ABA could increase anthocyanin synthesis under drought stress conditions potentially throughout microRNA156 (miRNA156).

Flowering plants undergo juvenile vegetative, adult vegetative, and reproductive phases. Lily plants ( Lilium spp.) develop scaly leaves during their juvenile vegetative phase. Stem elongation occurs in the adult vegetative phase and is followed by floral transition. As the duration of the juvenile vegetative phase is long in lilies, the microRNA156 (miR156) and SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL) modules are expected to play a major role in vegetative phase change and flower induction. In the present study, we aimed to explore the functions of lily SLP13A. We evaluated phenotypic changes and gene expression in L. formosanum plants overexpressing miR156-resistant SPL13A ( rSPL13A ) and examined the accumulation levels of gene transcripts and mature miRNAs in non-transformed L. longiflorum plants. Lily plants overexpressing rSPL13A exhibited stem elongation under non-inductive conditions, and FLOWERING LOCUS T ( FT ) genes were poorly involved in this stem elongation. Flowering was induced in the transformed plants with elongated stems, and the accumulation of MADS5 (APETALA1) transcripts and mature miR172 was elevated in these plants. In non-transformed lilies, SPL13A transcripts were highly accumulated in the shoot apices of both juvenile and adult plants. As mature miR156 was poorly accumulated in the shoot apices of the adult plants, SPL13A was active enough to stimulate stem elongation and flower induction. In contrast, mature miR156 was reliably detected in shoot apices of the juvenile plants. Because our transient assay using tobacco plants expressing a SPL13A-GFP fusion protein indicated that miR156 repressed SPL13A expression mainly at the translational level, SPL13A activity should be insufficient to stimulate stem elongation in the juvenile plants. In addition, the accumulation of MADS5 transcripts and mature miR172 in the shoot apices increased with plant growth and peaked before the transition to the reproductive phase. Therefore, we conclude that SPL13A regulates stem elongation in the adult vegetative phase, which differs from the mechanisms evaluated in Arabidopsis and rice, wherein stem elongation proceeds in a reproductive phase and FT genes are heavily involved in it, and that SPL13A induces flowering by the activation of genes related to the age pathway underlying floral transition, as APETALA1 and primary-MIR172 are mainly involved in this pathway.

Grasses possess basal and aerial axillary buds. Previous studies have largely focused on basal bud (tiller) formation but scarcely touched on aerial buds, which may lead to aerial branch development. Genotypes with and without aerial buds were identified in switchgrass (Panicum virgatum), a dedicated bioenergy crop. Bud development was characterized using scanning electron microscopy. Microarray, RNA-seq and quantitative reverse transcription polymerase chain reaction (RT-qPCR) were used to identify regulators of bud formation. Gene function was characterized by down-regulation and overexpression. Overexpression of miR156 induced aerial bud formation in switchgrass. Various analyses revealed that SQUAMOSA PROMOTER BINDING PROTEIN LIKE4 (SPL4), one of the miR156 targets, directly regulated aerial axillary bud initiation. Down-regulation of SPL4 promoted aerial bud formation and increased basal buds, while overexpression of SPL4 seriously suppressed bud formation and tillering. RNA-seq and RT-qPCR identified potential downstream genes of SPL4. Unlike all previously reported genes acting as activators of basal bud initiation, SPL4 acts as a suppressor for the formation of both aerial and basal buds. The miR156-SPL4 module predominantly regulates aerial bud initiation and partially controls basal bud formation. Genetic manipulation of SPL4 led to altered plant architecture with increased branching, enhanced regrowth after cutting and improved biomass yield.

Lettuce (Lactuca sativa) is a globally important leafy vegetable. Understanding the genetic factors underlying its growth and regeneration is critical for advancing agricultural productivity and biotechnological applications. To address this, the study aimed to comprehensively identify and characterize the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) gene family in lettuce and investigate their potential roles in plant development and regeneration. As a result, 22 SPL genes were identified within the lettuce genome. Fourteen of these genes contain recognition sites for microRNA156, suggesting post-transcriptional regulation. Each LsSPL protein has the highly conserved SBP domain and is predicted to localize in the nucleus. Analysis of public RNA-seq datasets revealed tissue-specific expression patterns of the 22 LsSPL genes, with five highly expressed in leaves, four in roots, and three in stems, indicating their distinct roles in plant development. Overexpression of lettuce miRNA156c (miR156-OX) led to reduced leaf size and delayed flowering time, whereas suppression of miR156 (miR156-STTM) resulted in increased leaf size. Surprisingly, cotyledon explants from miR156-OX lettuce lines exhibited a 1.9-fold increase in shoot regeneration compared to wild-type, whereas miR156-STTM lines exhibited a 54.3% decrease. This enhanced in vitro shoot regeneration was also observed in ectopic miR156-overexpression tomato lines, suggesting a conserved mechanism. Quantitative RT-PCR analysis confirmed the downregulation of LsSPL13A.1, LsSPL13A.2, and LsSPL12.2 in miR156-OX lines and their upregulation in miR156-STTM lines after 5 days of callus induction, implicating their specific roles in in vitro organogenesis and plant regeneration. This comprehensive analysis provides valuable insights into the SPL gene family and the miR156-SPLs regulatory network, specifically highlighting its role in regeneration. These findings hold the potential for improving plant growth, development, and biotechnological applications.

The juvenile-to-adult (JA) phase transition is an important event for optimizing vegetative growth and reproductive success in plants. Among the Poaceae crops, which are a vital food source for humans, studies of the JA phase transition have been restricted to rice and maize. We studied the morphological and genetic changes that occur during the early development of sorghum and found that dramatic changes occur in shoot architecture during the early vegetative stages. Changes were observed in leaf size, leaf shape, numbers of trichomes, and size of the shoot apical meristem. In particular, the length/width ratios of the leaf blades in the fifth and upper leaves were completely different from those of the second to fourth leaves. The fifth and upper leaves have trichomes on their adaxial sides, which were absent on the lower leaves. We also analyzed expression of two microRNAs that are known to be molecular markers of the JA phase transition and found that expression of miR156 was highest in the second to fourth leaves and then was gradually down-regulated, whereas miR172 expression followed the opposite pattern. These results suggest that in sorghum, the second and third leaves represent the juvenile phase, the fourth and fifth leaves are in the transition stage, and the sixth and upper leaves are in the adult phase. Thus, the JA phase transition occurs during the fourth and fifth leaf stages. These findings are expected to be useful for understanding the early development of sorghum.

MicroRNA156 (miR156) regulates a network of downstream genes to affect plant growth and development. We previously generated alfalfa ( Medicago sativa ) plants that overexpress homologous miR156 (MsmiR156OE), and identified three of its SPL target genes. These plants exhibited increased vegetative yield, delayed flowering and longer roots. In this study, we aimed to elucidate the effect of miR156 on the root system, including effect on nodulation and nitrogen fixation. We found that MsmiR156 overexpression increases root regeneration capacity in alfalfa, but with little effect on root biomass at the early stages of root development. MsmiR156 also promotes nitrogen fixation activity by upregulating expression of nitrogenase-related genes FixK , NifA and RpoH in roots inoculated with Sinorrhizobium meliloti. Furthermore, we conducted transcriptomics analysis of MsmiR156OE alfalfa roots and identified differentially expressed genes belonging to 132 different functional categories, including plant cell wall organization, peptidyl-hypusine synthesis, and response to water stress. Expression analysis also revealed miR156 effects on genes involved in nodulation, root development and phytohormone biosynthesis. The present findings suggest that miR156 regulates root development and nitrogen fixation activity. Taken together, these findings highlight the important role that miR156 may play as a tool in the biotechnological improvement of alfalfa, and potentially other crops.