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Summary The rice root system is important for growth. The crosstalk between auxin and cytokinin mediates root initiation and elongation. However, it remains unclear how the transcriptional network upstream of the auxin and cytokinin signalling pathways determines root development. Here, we observed that the knockdown of OsNAC2, which encodes a NAC transcription factor, increased the primary root length and the number of crown roots. OsNAC2 predominantly expressed in primary root tips, crown roots and lateral root primordia, implying it influences root development. Molecular analyses revealed that the expressions of auxin‐ and cytokinin‐responsive genes were affected in OsNAC2‐overexpressing (OsNAC2‐OX; ON7 and ON11), RNA interference (OsNAC2‐RNAi; RNAi25 and RNAi31) and CRISPR/Cas9 plants. Additionally, OsNAC2 can directly bind to the promoters of IAA inactivation‐related genes (GH3.6 and GH3.8), an IAA signalling‐related gene (OsARF25), and a cytokinin oxidase gene (OsCKX4). Furthermore, genetic analysis of ON11/osgh3.6 and RNAi31/osckx4 homozygote confirmed that OsCKX4 and OsGH3.6 functioned downstream of OsNAC2. The mRNA levels of CROWN ROOTLESS (CRL) genes and cyclin‐dependent protein kinase (CDK) genes increased in OsNAC2‐RNAi and OsNAC2‐cas9 lines while reduced in OsNAC2‐OX lines. Thus, we describe that OsNAC2 functions as an upstream integrator of auxin and cytokinin signals that affect CRL and CDK production to regulate cell division during root development. This novel auxin‐OsNAC2‐cytokinin model should provide a new insight into the understanding of NAC TFs and crosstalk of auxin and cytokinin pathway, and can be potentially applied in agriculture to enhance rice yields by genetic approaches.

Key messageWe reveal the onset and dynamic tissue-specific cytokinin signaling domains and functional importance of auxin in the auxin–cytokinin interaction domains in shaping root architecture in the economically important rice plant.Plant hormones such as auxin and cytokinin are central regulators of root organogenesis. Typical in the grass species, the root system in rice is primarily composed of post-embryonic adventitious/crown roots (ARs/CRs). Antagonistic auxin–cytokinin activities mutually balance each other to ensure proper root development. Cytokinin has been shown to inhibit crown root initiation in rice; albeit, the responsive domains remain elusive during the initiation and outgrowth of crown root primordia (CRP). Here, we show the cytokinin response domains during various stages of CRP development. RNA–RNA in situ hybridization and protein immunohistochemistry studies of the reporter gene expressed under the cytokinin responsive synthetic promoter revealed detailed spatio-temporal cytokinin signaling domains in the developing CRP. Furthermore, rice lines genetically depleted for endogenous auxin in the cytokinin responsive domains provided insight into the functional importance of auxin signaling during crown root development. Thus, our study demonstrates the onset and dynamic tissue-specific cytokinin response and functional significance of auxin–cytokinin interaction during root architecture formation in rice, a model grass species.

Main conclusionThis study reveals that the process of crown root development and auxin-induced de novo root organogenesis during in vitro plantlet regeneration share a common auxin-OsWOX10 regulatory module in rice.In the fibrous-type root system of rice, the crown roots (CR) are developed naturally from the shoot tissues. Generation of robust auxin response, followed by activation of downstream cell fate determinants and signaling pathways at the onset of crown root primordia (CRP) establishment is essential for new root initiation. During rice tissue culture, embryonic calli are induced to regenerate shoots in vitro which undergo de novo root organogenesis on an exogenous auxin-supplemented medium, but the mechanism underlying spatially restricted root organogenesis remains unknown. Here, we reveal the dynamics of progressive activation of genes involved in auxin homeostasis and signaling during initiation and outgrowth of rice crown root primordia. By comparative global dataset analysis, we identify the crown root primordia-expressed genes whose expression is also regulated by auxin signaling. In-depth spatio-temporal expression pattern analysis shows that the exogenous application of auxin induces a set of key transcription factors exclusively in the spatially positioned CRP. Further, functional analysis of rice WUSCHEL-RELATED HOMEOBOX 10 (OsWOX10) during in vitro plantlet regeneration from embryogenic calli shows that it promotes de novo root organogenesis from regenerated shoots. Expression of rice OsWOX10 also induces adventitious roots (AR) in Arabidopsis, independent of homologous endogenous Arabidopsis genes. Together, our findings reveal that a common auxin-transcription factor regulatory module is involved in root organogenesis under different conditions.

Background Waterlogging is a serious abiotic stress to plant growth because it results in the decline in the supplement of oxygen to submerged tissues. Although cucumber ( Cucumis sativus L.) is sensitive to waterlogging, its ability to generate adventitious roots (ARs) facilitates gas diffusion and increases plant survival when the oxygen concentration is decreased. To gain a better understanding of the molecular mechanisms that enable de novo AR primordia emergence upon waterlogging, the RNA sequencing-based transcriptomic responses of two contrasting cucumber genotypes, Zaoer-N (waterlogging tolerant) and Pepino (waterlogging sensitive), which differed in their abilities to form AR were compared. Results More than 27,000 transcripts were detected in cucumber hypocotyls, from which 1494 and 1766 genes in ‘Zaoer-N’ and ‘Pepino’, respectively, were differentially expressed 2 days after waterlogging. The significant positive correlation between RNA sequencing data and a qPCR analysis indicated that the identified genes were credible. A comparative analysis revealed that genes functioning in carbohydrate mobilization, nitrate assimilation, hormone production and signaling pathways, transcription factors and cell division might contribute to the waterlogging-triggered AR primordia initiation. Ethylene was determined to be an important plant hormone responsible for the cucumber ARs initiation. Additionally, genes encoding cytochrome P450, ankyrin repeat-containing proteins and sulfite oxidases were determined as important in waterlogging acclimation. Conclusion This research broadens our understanding of the mechanism underlying waterlogging-triggered ARs emergence, and provides valuable information for the breeding of cucumber with enhanced waterlogging tolerance.

• Cytokinin and auxin are key regulators of plant growth and development. During the last decade transport mechanisms have turned out to be the key for the control of local and longdistance hormone distributions. In contrast with auxin, cytokinin transport is poorly understood. • Here, we show that Arabidopsis thaliana AZG2, a member of the AZG purine transporter family, acts as cytokinin transporter involved in root system architecture determination. Even though purines are substrates for both AZG1 and AZG2, we found distinct transport mechanisms. • The expression of AZG2 is restricted to a small group of cells surrounding the lateral root (LR) primordia and induced by auxins. Compared to the wild-type (WT), mutants carrying loss-of-function alleles of AZG2 have higher LR density, suggesting that AZG2 is part of a regulatory pathway in LR emergence. Moreover, azg2 is partially insensitive to exogenous cytokinin, which is consistent with the observation that the cytokinin reporter TCSnpro:GFP showed lower fluorescence signal in the roots of azg2 compared to the WT. • These results indicate a defective cytokinin signalling pathway in the region of LR primordia. The integration of AZG2 subcellular localization and cytokinin transport capacity data allowed us to propose a local cytokinin : auxin signalling model for the regulation of LR emergence.

This review summarizes recent studies on lateral root formation that have begun to reveal detailed regulatory mechanisms at different developmental stages, with an emphasis on the multiple roles of auxin. Abstract Root systems can display variable architectures that contribute to survival strategies of plants. The model plant Arabidopsis thaliana possesses a tap root system, in which the primary root and lateral roots (LRs) are major architectural determinants. The phytohormone auxin fulfils multiple roles throughout LR development. In this review, we summarize recent advances in our understanding of four aspects of LR formation: (i) LR positioning, which determines the spatial distribution of lateral root primordia (LRP) and LRs along primary roots; (ii) LR initiation, encompassing the activation of nuclear migration in specified lateral root founder cells (LRFCs) up to the first asymmetric cell division; (iii) LR outgrowth, the 'primordium-intrinsic' patterning of de novo organ tissues and a meristem; and (iv) LR emergence, an interaction between LRP and overlaying tissues to allow passage through cell layers. We discuss how auxin signaling, embedded in a changing developmental context, plays important roles in all four phases. In addition, we discuss how rapid progress in gene network identification and analysis, modeling, and four-dimensional imaging techniques have led to an increasingly detailed understanding of the dynamic regulatory networks that control LR development.

Aerial architecture in higher plants is dependent on the activity of the shoot apical meristem (SAM) and axillary meristems (AMs). The SAM produces a main shoot and leaf primordia, while AMs are generated at the axils of leaf primordia and give rise to branches and flowers. Therefore, the formation of AMs is a critical step in the construction of plant architecture. Here, we characterized the rice (Oryza sativa) lax panicle2 (lax2) mutant, which has altered AM formation. LAX2 regulates the branching of the aboveground parts of a rice plant throughout plant development, except for the primary branch in the panicle. The lax2 mutant is similar to lax paniclei (Iax1) in that it lacks an AM in most of the lateral branching of the panicle and has a reduced number of AMs at the vegetative stage. The lax1 lax2 double mutant synergistically enhances the reduced-branching phenotype, indicating the presence of multiple pathways for branching. LAX2 encodes a nuclear protein that contains a plant-specific conserved domain and physically interacts with LAX1. We propose that LAX2 is a novel factor that acts together with LAX1 in rice to regulate the process of AM formation.

Summary Adventitious roots occur naturally in many species and can also be induced from explants of some tree species including Populus, providing an important means of clonal propagation. Auxin has been identified as playing a crucial role in adventitious root formation, but the associated molecular regulatory mechanisms need to be elucidated. In this study, we examined the role of PagFBL1, the hybrid poplar (Populus alba × P. glandulosa clone 84K) homolog of Arabidopsis auxin receptor TIR1, in adventitious root formation in poplar. Similar to the distribution pattern of auxin during initiation of adventitious roots, PagFBL1 expression was concentrated in the cambium and secondary phloem in stems during adventitious root induction and initiation phases, but decreased in emerging adventitious root primordia. Overexpressing PagFBL1 stimulated adventitious root formation and increased root biomass, while knock‐down of PagFBL1 transcript levels delayed adventitious root formation and decreased root biomass. Transcriptome analyses of PagFBL1 overexpressing lines indicated that an extensive remodelling of gene expression was stimulated by auxin signalling pathway during early adventitious root formation. In addition, PagIAA28 was identified as downstream targets of PagFBL1. We propose that the PagFBL1‐PagIAA28 module promotes adventitious rooting and could be targeted to improve Populus propagation by cuttings.

• VERNALIZATION2 (VRN2), an angiosperm-specific subunit of the polycomb repressive complex 2 (PRC2), is an oxygen (O₂)-regulated target of the PCO branch of the PRT6 N-degron pathway of ubiquitin-mediated proteolysis. How this post-translational regulation coordinates VRN2 activity remains to be fully established. • Here we use Arabidopsis thaliana ecotypes, mutants and transgenic lines to determine how control of VRN2 stability contributes to its functions during plant development. • VRN2 localizes to endogenous hypoxic regions in aerial and root tissues. In the shoot apex, VRN2 differentially modulates flowering time dependent on photoperiod, whilst its presence in lateral root primordia and the root apical meristem negatively regulates root system architecture. Ectopic accumulation of VRN2 does not enhance its effects on flowering, but does potentiate its repressive effects on root growth. In late-flowering vernalization-dependent ecotypes, VRN2 is only active outside meristems when its proteolysis is inhibited in response to cold exposure, as its function requires concomitant cold-triggered increases in other PRC2 subunits and cofactors. • We conclude that the O₂-sensitive N-degron of VRN2 has a dual function, confining VRN2 to meristems and primordia, where it has specific developmental roles, whilst also permitting broad accumulation outside of meristems in response to environmental cues, leading to other functions.

Nitrogen fixation in soybean takes place in root nodules that arise from de novo cell divisions in the root cortex. Although several early nodulin genes have been identified, the mechanism behind the stimulation of cortical cell division during nodulation has not been fully resolved. Here we provide evidence that two paralogs of soybean SHORT-ROOT (GmSHR) play vital roles in soybean nodulation. Expression of GmSHR4 and GmSHR5 (GmSHR4/5) is induced in cortical cells at the beginning of nodulation, when the first cell divisions occur. The expression level of GmSHR4/5 is positively associated with cortical cell division and nodulation. Knockdown of GmSHR5 inhibits cell division in outer cortical layers during nodulation. Knockdown of both paralogs disrupts the cell division throughout the cortex, resulting in poorly organized nodule primordia with delayed vascular tissue formation. GmSHR4/5 function by enhancing cytokinin signaling and activating early nodulin genes. Interestingly, D-type cyclins act downstream of GmSHR4/5, and GmSHR4/5 form a feedforward loop regulating D-type cyclins. Overexpression of D-type cyclins in soybean roots also enhanced nodulation. Collectively, we conclude that the GmSHR4/5-mediated pathway represents a vital module that triggers cytokinin signaling and activates D-type cyclins during nodulation in soybean.