WUSCHEL-Related Homeobox Genes Cooperate with Cytokinin Signalling to Promote Bulbil Formation in Lilium lancifolium

The bulbil is an important vegetative reproductive organ in triploid Lilium lancifolium. Based on our previously obtained transcriptome data, we screened two WUSCHCEL-related homeobox (WOX) genes closely related to bulbil formation, LlWOX9 and LlWOX11. However, the biological functions and regulatory mechanisms of LlWOX9 and LlWOX11 are unclear. In this study, we cloned the full-length coding sequences of LlWOX9 and LlWOX11. Transgenic Arabidopsis showed increased branch numbers, and the overexpression of LlWOX9 and LlWOX11 in stem segments promoted bulbil formation, while the silencing of LlWOX9 and LlWOX11 inhibited bulbil formation, indicating that LlWOX9 and LlWOX11 are positive regulators of bulbil formation. Cytokinins acting through type-B response regulators (type-B RRs) could bind to the promoters of LlWOX9 and LlWOX11 and promote their transcription. LlWOX11 could enhance cytokinin pathway signalling by inhibiting the transcription of type-A LlRR9. Our study enriches the understanding of the regulation of plant development by the WOX gene family and lays a foundation for further research on the molecular mechanism of bulbil formation in lily.

Genome-Wide Identification and Expression Profiling Analysis of WOX Family Protein-Encoded Genes in Triticeae Species

The WOX family is a group of plant-specific transcription factors which regulate plant growth and development, cell division and differentiation. From the available genome sequence databases of nine Triticeae species, 199 putative WOX genes were identified. Most of the identified WOX genes were distributed on the chromosomes of homeologous groups 1 to 5 and originated via the orthologous evolution approach. Parts of WOX genes in Triticum aestivum were confirmed by the specific PCR markers using a set of Triticum. durum-T. aestivum genome D substitution lines. All of these identified WOX proteins could be grouped into three clades, similar to those in rice and Arabidopsis. WOX family members were conserved among these Triticeae plants; all of them contained the HOX DNA-binding homeodomain, and WUS clade members contained the characteristic WUS-box motif, while only WUS and WOX9 contained the EAR motif. The RNA-seq and qPCR analysis revealed that the TaWOX genes had tissue-specific expression feature. From the expression patterns of TaWOX genes during immature embryo callus production, TaWOX9 is likely closely related with the regulation of regeneration process in T. aestivum. The findings in this study could provide a basis for evolution and functional investigation and practical application of the WOX family genes in Triticeae species.

Genome-Wide Identification and Characterization of WOX Genes in Cucumis sativus

WUSCHEL-related homeobox (WOX) proteins are plant-specific transcription factors that are profoundly involved in regulation of plant development and stress responses. In this study, we totally identified 11 WOX transcription factor family members in cucumber (Cucumis sativus, CsWOXs) genome and classified them into three clades with nine subclades based on phylogenetic analysis results. Alignment of amino acid sequences revealed that all WOX members in cucumber contained the typical homeodomain, which consists of 60-66 amino acids and is folded into a helix-turn-helix structure. Gene duplication event analysis indicated that CsWOX1a and CsWOX1b were a segment duplication pair, which might affect the number of WOX members in cucumber genome. The expression profiles of CsWOX genes in different tissues demonstrated that the members sorted into the ancient clade (CsWOX13a and CsWOX13b) were constitutively expressed at higher levels in comparison to the others. Cis-element analysis in promoter regions suggested that the expression of CsWOX genes was associated with phytohormone pathways and stress responses, which was further supported by RNA-seq data. Taken together, our results provide new insights into the evolution of cucumber WOX genes and improve our understanding about the biological functions of CsWOX family.

The Molecular Basis of Age-Modulated Plant De Novo Root Regeneration Decline in Arabidopsis thaliana

Plants possess a regeneration capacity that enables them to survive after wounding. For example, detached Arabidopsis thaliana leaves are able to form adventitious roots from their cutting sites even in the absence of exogenous hormone supplements, as process termed de novo root regeneration (DNRR). Wounding rapidly induces auxin biosynthesis at the cutting sites and then elicits a signaling cascade to promote cell fate transitions and finally generate the adventitious roots. However, rooting rates in older plants are much lower than in younger leaf explants. In this review, we highlight the recent breakthroughs in the understanding of DNRR decay in older plants from at least two independent signaling routes: (i) via the accumulation of EIN3 protein in older plants, which directly suppresses expression of WUSCHEL RELATED HOMEOBOX (WOX) genes to inhibit rooting; (ii) the miR156-SPLs-AP2/ERFs pathway, which modulates root regeneration by reducing auxin biosynthesis.

WOX genes expression during the formation of new lateral roots from secondary structures in Populus nigra (L.) taproot

Despite the large amounts of data available on lateral root formation, little is known about their initiation from secondary structures. In the present work, we applied a bending treatment to Populus nigra (L.) woody taproots to induce the formation of new lateral roots. The development of lateral roots was monitored by stereomicroscopic examination of cross-sections. Tissues were sampled from the bending zone in the proximity of the vascular cambium before (time 0) and after the application of bending at three different time points (24, 48, and 72 h) and analyzed for the expression of P. nigra WOX homologs. The initiation of new lateral roots was observed to originate from the vascular cambium zone and was followed by primordium formation and root emergence. PnWOX4a, PnWOX4b, PnWOX5a, PnWOX5b, PnWOX11/12a, and PnWOX11/12b were shown to be expressed during the formation of new lateral roots at different developmental stages. The mechanical stress simulated by bending treatment was shown to activate the molecular mechanism leading to the expression of WOX genes, which are hypothesized to control SLR formation in the cambium zone of poplar taproot.

Molecular Evolution of Auxin-Mediated Root Initiation in Plants

The root originated independently in euphyllophytes (ferns and seed plants) and lycophytes; however, the molecular evolutionary route of root initiation remains elusive. By analyses of the fern Ceratopteris richardii and seed plants, here we show that the molecular pathway involving auxin, intermediate-clade WUSCHEL-RELATED HOMEOBOX (IC-WOX) genes, and WUSCHEL-clade WOX (WC-WOX) genes could be conserved in root initiation. We propose that the “auxin>IC-WOX>WC-WOX” module in root initiation might have arisen in the common ancestor of euphyllophytes during the second origin of roots, and that this module has further developed during the evolution of different root types in ferns and seed plants.

WOX9 functions antagonistic to STF and LAM1 to regulate leaf blade expansion in Medicago truncatula and Nicotiana sylvestris

Plant specific WOX family transcription factors are known to regulate embryogenesis, meristem maintenance and lateral organ development. Modern clade WOX genes function through a transcriptional repression mechanism, and the intermediate clade transcriptional activator WOX9 functions with the repressor WOX genes in embryogenesis and meristems maintenance, but the mechanism of this interaction is unclear. WOX1 homologues STF and LAM1 are required for leaf blade outgrowth in Medicago truncatula and Nicotiana Sylvestris, respectively. Here we show that WOX9 negatively regulates leaf blade outgrowth and functions antagonistically to STF and LAM1. While NsWOX9 ectopic expression enhances the lam1 mutant phenotype, and antisense expression partially rescues the lam1 mutant, both overexpression of NsWOX9 and knockout by CRISPR/Cas9 genome editing in N. sylvestris resulted in a range of severe leaf blade distortions, indicating that controlled negative regulation by NsWOX9 is required for proper blade development. Our results indicate that direct repression of WOX9 transcriptional activation activity by the transcriptional repressor STF/LAM1 is required for correct blade architecture and patterning in M. truncatula and N. sylvestris. These findings suggest that a balance between transcriptional activation and repression mechanisms by direct interaction of activator and repressor WOX genes may be required for cell proliferation and differentiation homeostasis, and could be an evolutionarily conserved mechanism for the development of complex and diverse morphology in higher plants.One sentence summaryWOX9 negatively regulates blade outgrowth antagonizing STF function but directly repressed by STF indicating WOX-mediated homeostasis in cell proliferation and differentiation during leaf morphogenesis.