Genetics of Shoot Meristem and Shoot Regeneration.

Plants exhibit remarkable lineage plasticity, allowing them to regenerate organs that differ from their respective origins. Such developmental plasticity is dependent on the activity of pluripotent founder cells or stem cells residing in meristems. At the shoot apical meristem (SAM), the constant flow of cells requires continuing cell specification governed by a complex genetic network, with the WUSCHEL transcription factor and phytohormone cytokinin at its core. In this review, I discuss some intriguing recent discoveries that expose new principles and mechanisms of patterning and cell specification acting both at the SAM and prior to meristem organogenesis during shoot regeneration. I also highlight unanswered questions and future challenges in the study of SAM and meristem regeneration. Finally, I put forward a model describing stochastic events mediated by epigenetic factors to explain how the gene regulatory network might be initiated at the onset of shoot regeneration.

Heterogeneous identity, stiffness and growth characterise the shoot apex of Arabidopsis stem cell mutants.

Stem cell homeostasis in the shoot apical meristem involves a core regulatory feedback loop between the signalling peptide CLAVATA3 (CLV3), produced in stem cells, and the transcription factor WUSCHEL, expressed in the underlying organising centre. clv3 mutant meristems display massive overgrowth, which is thought to be caused by stem cell overproliferation, although it is unknown how uncontrolled stem cell divisions lead to this altered morphology. Here, we reveal local buckling defects in mutant meristems, and use analytical models to show how mechanical properties and growth rates may contribute to the phenotype. Indeed, clv3 mutant meristems are mechanically more heterogeneous than the wild type, and also display regional growth heterogeneities. Furthermore, stereotypical wild-type meristem organisation, in which cells simultaneously express distinct fate markers, is lost in mutants. Finally, cells in mutant meristems are auxin responsive, suggesting that they are functionally distinguishable from wild-type stem cells. Thus, all benchmarks show that clv3 mutant meristem cells are different from wild-type stem cells, suggesting that overgrowth is caused by the disruption of a more complex regulatory framework that maintains distinct genetic and functional domains in the meristem.

An updated model of shoot apical meristem regulation by ERECTA family and CLAVATA3 signaling pathways in Arabidopsis.

The shoot apical meristem (SAM) gives rise to the aboveground organs of plants. The size of the SAM is relatively constant due to the balance between stem cell replenishment and cell recruitment into new organs. In angiosperms, the transcription factor WUSCHEL (WUS) promotes stem cell proliferation in the central zone of the SAM. WUS forms a negative feedback loop with a signaling pathway activated by CLAVATA3 (CLV3). In the periphery of the SAM, the ERECTA family receptors (ERfs) constrain WUS and CLV3 expression. Here, we show that four ligands of ERfs redundantly inhibit the expression of these two genes. Transcriptome analysis confirmed that WUS and CLV3 are the main targets of ERf signaling and uncovered new ones. Analysis of promoter reporters indicated that the WUS expression domain mostly overlaps with the CLV3 domain and does not shift along the apical-basal axis in clv3 mutants. Our three-dimensional mathematical model captured gene expression distributions at the single-cell level under various perturbed conditions. Based on our findings, CLV3 regulates cellular levels of WUS mostly through autocrine signaling, and ERfs regulate the spatial expression of WUS, preventing its encroachment into the peripheral zone.

Root stem cell niche organizer specification by molecular convergence of PLETHORA and SCARECROW transcription factor modules.

Continuous formation of somatic tissues in plants requires functional stem cell niches where undifferentiated cells are maintained. In Arabidopsis thaliana, PLETHORA (PLT) and SCARECROW (SCR) genes are outputs of apical-basal and radial patterning systems, and both are required for root stem cell specification and maintenance. The WUSCHEL-RELATED HOMEOBOX 5 (WOX5) gene is specifically expressed in and required for functions of a small group of root stem cell organizer cells, also called the quiescent center (QC). PLT and SCR are required for QC function, and their expression overlaps in the QC; however, how they specify the organizer has remained unknown. We show that PLT and SCR genetically and physically interact with plant-specific teosinte-branched cycloidea PCNA (TCP) transcription factors to specify the stem cell niche during embryogenesis and maintain organizer cells post-embryonically. PLT-TCP-SCR complexes converge on PLT-binding sites in the WOX5 promoter to induce expression.

Pivotal role of STIP in ovule pattern formation and female germline development in Arabidopsis thaliana.

In spermatophytes the sporophytic (diploid) and the gametophytic (haploid) generations co-exist in ovules, and the coordination of their developmental programs is of pivotal importance for plant reproduction. To achieve efficient fertilization, the haploid female gametophyte and the diploid ovule structures must coordinate their development to form a functional and correctly shaped ovule. WUSCHEL-RELATED HOMEOBOX (WOX) genes encode a family of transcription factors that share important roles in a wide range of processes throughout plant development. Here, we show that STIP is required for the correct patterning and curvature of the ovule in Arabidopsis thaliana. The knockout mutant stip-2 is characterized by a radialized ovule phenotype due to severe defects in outer integument development. In addition, alteration of STIP expression affects the correct differentiation and progression of the female germline. Finally, our results reveal that STIP is required to tightly regulate the key ovule factors INNER NO OUTER, PHABULOSA and WUSCHEL, and they define a novel genetic interplay in the regulatory networks determining ovule development.

Evolution of sex-determination in dioecious plants: From active Y to X/A balance?

Sex chromosomes in plants have been known for a century, but only recently have we begun to understand the mechanisms behind sex determination in dioecious plants. Here, we discuss evolution of sex determination, focusing on Silene latifolia, where evolution of separate sexes is consistent with the classic "two mutations" model-a loss of function male sterility mutation and a gain of function gynoecium suppression mutation, which turned an ancestral hermaphroditic population into separate males and females. Interestingly, the gynoecium suppression function in S. latifolia evolved via loss of function in at least two sex-linked genes and works via gene dosage balance between sex-linked, and autosomal genes. This system resembles X/A-ratio-based sex determination systems in Drosophila and Rumex, and could represent a steppingstone in the evolution of X/A-ratio-based sex determination from an active Y system.

WUSCHEL-related homeobox family genes in rice control lateral root primordium size.

The development of a plastic root system is essential for stable crop production under variable environments. Rice plants have two types of lateral roots (LRs): S-type (short and thin) and L-type (long, thick, and capable of further branching). LR types are determined at the primordium stage, with a larger primordium size in L-types than S-types. Despite the importance of LR types for rice adaptability to variable water conditions, molecular mechanisms underlying the primordium size control of LRs are unknown. Here, we show that two WUSCHEL-related homeobox (WOX) genes have opposing roles in controlling LR primordium (LRP) size in rice. Root tip excision on seminal roots induced L-type LR formation with wider primordia formed from an early developmental stage. QHB/OsWOX5 was isolated as a causative gene of a mutant that is defective in S-type LR formation but produces more L-type LRs than wild-type (WT) plants following root tip excision. A transcriptome analysis revealed that OsWOX10 is highly up-regulated in L-type LRPs. OsWOX10 overexpression in LRPs increased the LR diameter in an expression-dependent manner. Conversely, the mutation in OsWOX10 decreased the L-type LR diameter under mild drought conditions. The qhb mutants had higher OsWOX10 expression than WT after root tip excision. A yeast one-hybrid assay revealed that the transcriptional repressive activity of QHB was lost in qhb mutants. An electrophoresis mobility shift assay revealed that OsWOX10 is a potential target of QHB. These data suggest that QHB represses LR diameter increase, repressing OsWOX10 Our findings could help improve root system plasticity under variable environments.

A new chromosome-scale genome of wild Brassica oleracea provides insights into the domestication of Brassica crops.

The cultivated diploid Brassica oleracea is an important vegetable crop, but the genetic basis of its domestication remains largely unclear in the absence of high-quality reference genomes of wild B. oleracea. Here, we report the first chromosome-level assembly of the wild Brassica oleracea L. W03 genome (total genome size, 630.7 Mb; scaffold N50, 64.6 Mb). Using the newly assembled W03 genome, we constructed a gene-based B. oleracea pangenome and identified 29 744 core genes, 23 306 dispensable genes, and 1896 private genes. We re-sequenced 53 accessions, representing six potential wild B. oleracea progenitor species. The results of the population genomic analysis showed that the wild B. oleracea populations had the highest level of diversity and represents the most closely related population to modern-day horticultural B. oleracea. In addition, the WUSCHEL gene was found to play a decisive role in domestication and to be involved in cauliflower and broccoli curd formation. We also illustrate the loss of disease-resistance genes during selection for domestication. Our results provide new insights into the domestication of B. oleracea and will facilitate the future genetic improvement of Brassica crops.

Global Analysis of the WOX Transcription Factor Family in Akebia trifoliata.

Akebia trifoliata is an economically important, self-incompatible fruit tree in the Lardizabalaceae family. Asexual propagation is the main strategy used to maintain excellent agronomic traits. However, the generation of adventitious roots during asexual propagation is very difficult. To study the important role of the WUSCHEL-related homeobox (WOX) transcription factor in adventitious root growth and development, we characterized this transcription factor family in the whole genome of A. trifoliata. A total of 10 AktWOXs were identified, with the following characteristics: length (657~11,328 bp), exon number (2~5), isoelectric point (5.65~9.03), amino acid number (176~361 AA) and molecular weight (20.500~40.173 kDa), and their corresponding expression sequence could also be detectable in the public transcriptomic data for A. trifoliata fruit. A total of 10 AktWOXs were classified into modern (6), intermediate (2) and ancient clades (2) and all AktWOXs had undergone strong purifying selection during evolution. The expression profile of AktWOXs during A. trifoliata adventitious root formation indicated that AktWOXs play an important role in the regulation of adventitious root development. Overall, this is the first study to identify and characterize the WOX family in A. trifoliata and will be helpful for further research on A. trifoliata adventitious root formation.

Identification of WUSCHEL-related homeobox gene and truncated small peptides in transformation efficiency improvement in Eucalyptus.

BACKGROUND: The WUSCHEL-related Homeobox (WOX) genes, which encode plant-specific homeobox (HB) transcription factors, play crucial roles in regulating plant growth and development. However, the functions of WOX genes are little known in Eucalyptus, one of the fastest-growing tree resources with considerable widespread cultivation worldwide. RESULTS: A total of nine WOX genes named EgWOX1-EgWOX9 were retrieved and designated from Eucalyptus grandis. From the three divided clades marked as Modern/WUS, Intermediate and Ancient, the largest group Modern/WUS (6 EgWOXs) contains a specific domain with 8 amino acids: TLQLFPLR. The collinearity, cis-regulatory elements, protein-protein interaction network and gene expression analysis reveal that the WUS proteins in E. grandis involve in regulating meristems development and regeneration. Furthermore, by externally adding of truncated peptides isolated from WUS specific domain, the transformation efficiency in E. urophylla × E. grandis DH32-29 was significant enhanced. The transcriptomics data further reveals that the use of small peptides activates metabolism pathways such as starch and sucrose metabolism, phenylpropanoid biosynthesis and flavonoid biosynthesis. CONCLUSIONS: Peptides isolated from WUS protein can be utilized to enhance the transformation efficiency in Eucalyptus, thereby contributing to the high-efficiency breeding of Eucalyptus.

A key to totipotency: Wuschel-like homeobox 2a unlocks embryogenic culture response in maize (Zea mays L.).

The ability of plant somatic cells to dedifferentiate, form somatic embryos and regenerate whole plants in vitro has been harnessed for both clonal propagation and as a key component of plant genetic engineering systems. Embryogenic culture response is significantly limited, however, by plant genotype in most species. This impedes advancements in both plant transformation-based functional genomics research and crop improvement efforts. We utilized natural variation among maize inbred lines to genetically map somatic embryo generation potential in tissue culture and identify candidate genes underlying totipotency. Using a series of maize lines derived from crosses involving the culturable parent A188 and the non-responsive parent B73, we identified a region on chromosome 3 associated with embryogenic culture response and focused on three candidate genes within the region based on genetic position and expression pattern. Two candidate genes showed no effect when ectopically expressed in B73, but the gene Wox2a was found to induce somatic embryogenesis and embryogenic callus proliferation. Transgenic B73 cells with strong constitutive expression of the B73 and A188 coding sequences of Wox2a were found to produce somatic embryos at similar frequencies, demonstrating that sufficient expression of either allele could rescue the embryogenic culture phenotype. Transgenic B73 plants were regenerated from the somatic embryos without chemical selection and no pleiotropic effects were observed in the Wox2a overexpression lines in the regenerated T0 plants or in the two independent events which produced T1 progeny. In addition to linking natural variation in tissue culture response to Wox2a, our data support the utility of Wox2a in enabling transformation of recalcitrant genotypes.

miR394 enhances WUSCHEL-induced somatic embryogenesis in Arabidopsis thaliana.

Many plant species can give rise to embryos from somatic cells after a simple hormone treatment, illustrating the remarkable developmental plasticity of differentiated plant cells. However, many species are recalcitrant to somatic embryo formation for unknown reasons, which poses a significant challenge to agriculture, where somatic embryogenesis is an important tool to propagate desired genotypes. The micro-RNA394 (miR394) promotes shoot meristem maintenance in Arabidopsis thaliana, but the underlying mechanisms have remained elusive. We analyzed whether miR394 affects indirect somatic embryogenesis and determined the transcriptome of embryogenic callus upon miR394-enhanced somatic embryogenesis. We show that ectopic miR394 expression enhances somatic embryogenesis in the recalcitrant Ler accession when co-expressed with the transcription factor WUSCHEL (WUS) and that miR394 acts in this process through silencing the target LEAF CURLING RESPONSIVENESS (LCR). Furthermore, we show that higher endogenous miR394 levels are required for the elevated embryogenic potential of the Columbia accession compared with Ler, providing a mechanistic explanation for this natural variation. Our transcriptional analysis provides a framework for miR394 function in regulating pluripotency by expanding WUS-mediated direct transcriptional repression.

ENO regulates tomato fruit size through the floral meristem development network.

A dramatic evolution of fruit size has accompanied the domestication and improvement of fruit-bearing crop species. In tomato (Solanum lycopersicum), naturally occurring cis-regulatory mutations in the genes of the CLAVATA-WUSCHEL signaling pathway have led to a significant increase in fruit size generating enlarged meristems that lead to flowers with extra organs and bigger fruits. In this work, by combining mapping-by-sequencing and CRISPR/Cas9 genome editing methods, we isolated EXCESSIVE NUMBER OF FLORAL ORGANS (ENO), an AP2/ERF transcription factor which regulates floral meristem activity. Thus, the ENO gene mutation gives rise to plants that yield larger multilocular fruits due to an increased size of the floral meristem. Genetic analyses indicate that eno exhibits synergistic effects with mutations at the LOCULE NUMBER (encoding SlWUS) and FASCIATED (encoding SlCLV3) loci, two central players in the evolution of fruit size in the domestication of cultivated tomatoes. Our findings reveal that an eno mutation causes a substantial expansion of SlWUS expression domains in a flower-specific manner. In vitro binding results show that ENO is able to interact with the GGC-box cis-regulatory element within the SlWUS promoter region, suggesting that ENO directly regulates SlWUS expression domains to maintain floral stem-cell homeostasis. Furthermore, the study of natural allelic variation of the ENO locus proved that a cis-regulatory mutation in the promoter of ENO had been targeted by positive selection during the domestication process, setting up the background for significant increases in fruit locule number and fruit size in modern tomatoes.

Transcriptomic and Metabolomic Investigation on Leaf Necrosis Induced by ZmWus2 Transient Overexpression in Nicotiana benthamiana.

WUSCHEL (WUS) is a crucial transcription factor in regulating plant stem cell development, and its expression can also improve genetic transformation. However, the ectopic expression of WUS always causes pleiotropic effects during genetic transformation, making it important to understand the regulatory mechanisms underlying these phenomena. In our study, we found that the transient expression of the maize WUS ortholog ZmWus2 caused severe leaf necrosis in Nicotiana benthamiana. We performed transcriptomic and non-target metabolomic analyses on tobacco leaves during healthy to wilted states after ZmWus2 transient overexpression. Transcriptomic analysis revealed that ZmWus2 transformation caused active metabolism of inositol trisphosphate and glycerol-3-phosphate, while also upregulating plant hormone signaling and downregulating photosystem and protein folding pathways. Metabolomic analysis mainly identified changes in the synthesis of phenylpropanoid compounds and various lipid classes, including steroid synthesis. In addition, transcription factors such as ethylene-responsive factors (ERFs), the basic helix-loop-helix (bHLH) factors, and MYBs were found to be regulated by ZmWus2. By integrating these findings, we developed a WUS regulatory model that includes plant hormone accumulation, stress responses, lipid remodeling, and leaf necrosis. Our study sheds light on the mechanisms underlying WUS ectopic expression causing leaf necrosis and may inform the development of future genetic transformation strategies.

Genome-Wide Identification and Characterization Analysis of WUSCHEL-Related Homeobox Family in Melon (Cucumis melo L.).

WUSCHEL-related homeobox (WOX) proteins are very important in controlling plant development and stress responses. However, the WOX family members and their role in response to abiotic stresses are largely unknown in melon (Cucumis melo L.). In this study, 11 WOX (CmWOX) transcript factors with conserved WUS and homeobox motif were identified and characterized, and subdivided into modern clade, ancient clade and intermediate clade based on bioinformatic and phylogenetic analysis. Evolutionary analysis revealed that the CmWOX family showed protein variations in Arabidopsis, tomato, cucumber, melon and rice. Alignment of protein sequences uncovered that all CmWOXs had the typical homeodomain, which consisted of conserved amino acids. Cis-element analysis showed that CmWOX genes may response to abiotic stress. RNA-seq and qRT-PCR results further revealed that the expression of partially CmWOX genes are associated with cold and drought. CmWOX13a and CmWOX13b were constitutively expressed under abiotic stresses, CmWOX4 may play a role in abiotic processes during plant development. Taken together, this study offers new perspectives on the CmWOX family's interaction and provides the framework for research on the molecular functions of CmWOX genes.

Long Non-Coding RNA lncWOX11a Suppresses Adventitious Root Formation of Poplar by Regulating the Expression of PeWOX11a.

Long non-coding RNAs (lncRNAs), a class of poorly conserved transcripts without protein-encoding ability, are widely involved in plant organogenesis and stress responses by mediating the transmission and expression of genetic information at the transcriptional, posttranscriptional, and epigenetic levels. Here, we cloned and characterized a novel lncRNA molecule through sequence alignment, Sanger sequencing, transient expression in protoplasts, and genetic transformation in poplar. lncWOX11a is a 215 bp transcript located on poplar chromosome 13, ~50 kbp upstream of PeWOX11a on the reverse strand, and the lncRNA may fold into a series of complex stem-loop structures. Despite the small open reading frame (sORF) of 51 bp within lncWOX11a, bioinformatics analysis and protoplast transfection revealed that lncWOX11a has no protein-coding ability. The overexpression of lncWOX11a led to a decrease in the quantity of adventitious roots on the cuttings of transgenic poplars. Further, cis-regulatory module prediction and CRISPR/Cas9 knockout experiments with poplar protoplasts demonstrated that lncWOX11a acts as a negative regulator of adventitious rooting by downregulating the WUSCHEL-related homeobox gene WOX11, which is supposed to activate adventitious root development in plants. Collectively, our findings imply that lncWOX11a is essential for modulating the formation and development of adventitious roots.

Paf1c defects challenge the robustness of flower meristem termination in Arabidopsis thaliana.

Although accumulating evidence suggests that gene regulation is highly stochastic, genetic screens have successfully uncovered master developmental regulators, questioning the relationship between transcriptional noise and intrinsic robustness of development. To identify developmental modules that are more or less resilient to large-scale genetic perturbations, we used the Arabidopsis polymerase II-associated factor 1 complex (Paf1c) mutant vip3, which is impaired in several RNA polymerase II-dependent transcriptional processes. We found that the control of flower termination was not as robust as classically pictured. In angiosperms, the floral female organs, called carpels, display determinate growth: their development requires the arrest of stem cell maintenance. In vip3 mutant flowers, carpels displayed a highly variable morphology, with different degrees of indeterminacy defects up to wild-type size inflorescence emerging from carpels. This phenotype was associated with variable expression of two key regulators of flower termination and stem cell maintenance in flowers, WUSCHEL and AGAMOUS The phenotype was also dependent on growth conditions. Together, these results highlight the surprisingly plastic nature of stem cell maintenance in plants and its dependence on Paf1c.

CRISPR/Cas9-mediated tetra-allelic mutation of the 'Green Revolution' SEMIDWARF-1 (SD-1) gene confers lodging resistance in tef (Eragrostis tef).

Tef is a staple food and a valuable cash crop for millions of people in Ethiopia. Lodging is a major limitation to tef production, and for decades, the development of lodging resistant varieties proved difficult with conventional breeding approaches. We used CRISPR/Cas9 to introduce knockout mutations in the tef orthologue of the rice SEMIDWARF-1 (SD-1) gene to confer semidwarfism and ultimately lodging resistance. High frequency recovery of transgenic and SD-1 edited tef lines was achieved in two tef cultivars by Agrobacterium-mediated delivery into young leaf explants of gene editing reagents along with transformation and regeneration enhancing morphogenic genes, BABY BOOM (BBM) and WUSCHEL2 (WUS2). All of the 23 lines analyzed by next-generation sequencing had at least two or more alleles of SD-1 mutated. Of these, 83% had tetra-allelic frameshift mutations in the SD-1 gene in primary tef regenerants, which were inherited in subsequent generations. Phenotypic data generated on T1 and T2 generations revealed that the sd-1 lines have reduced culm and internode lengths with no reduction in either panicle or peduncle lengths. These characteristics are comparable with rice sd-1 plants. Measurements of lodging, in greenhouse-grown plants, showed that sd-1 lines have significantly higher resistance to lodging at the heading stage compared with the controls. This is the first demonstration of the feasibility of high frequency genetic transformation and CRISPR/Cas9-mediated genome editing in this highly valuable but neglected crop. The findings reported here highlight the potential of genome editing for the improvement of lodging resistance and other important traits in tef.

Tomato CRABS CLAW paralogues interact with chromatin remodelling factors to mediate carpel development and floral determinacy.

CRABS CLAW (CRC) orthologues play a crucial role in floral meristem (FM) determinacy and gynoecium formation across angiosperms, the key developmental processes for ensuring successful plant reproduction and crop production. However, the mechanisms behind CRC mediated FM termination are far from fully understood. Here, we addressed the functional characterization of tomato (Solanum lycopersicum) paralogous CRC genes. Using mapping-by-sequencing, RNA interference and CRISPR/Cas9 techniques, expression analyses, protein-protein interaction assays and Arabidopsis complementation experiments, we examined their potential roles in FM determinacy and carpel formation. We revealed that the incomplete penetrance and variable expressivity of the indeterminate carpel-inside-carpel phenotype observed in fruit iterative growth (fig) mutant plants are due to the lack of function of the S. lycopersicum CRC homologue SlCRCa. Furthermore, a detailed functional analysis of tomato CRC paralogues, SlCRCa and SlCRCb, allowed us to propose that they operate as positive regulators of FM determinacy by acting in a compensatory and partially redundant manner to safeguard the proper formation of flowers and fruits. Our results uncover for the first time the physical interaction of putative CRC orthologues with members of the chromatin remodelling complex that epigenetically represses WUSCHEL expression through histone deacetylation to ensure the proper termination of floral stem cell activity.

Role for the shoot apical meristem in the specification of juvenile leaf identity in Arabidopsis.

The extent to which the shoot apical meristem (SAM) controls developmental decisions, rather than interpreting them, is a longstanding issue in plant development. Previous work suggests that vegetative phase change is regulated by signals intrinsic and extrinsic to the SAM, but the relative importance of these signals for this process is unknown. We investigated this question by examining the effect of meristem-deficient mutations on vegetative phase change and on the expression of key regulators of this process, miR156 and its targets, SPL transcription factors. We found that the precocious phenotypes of meristem-deficient mutants are a consequence of reduced miR156 accumulation. Tissue-specific manipulation of miR156 levels revealed that the SAM functions as an essential pool of miR156 early in shoot development, but that its effect on leaf identity declines with age. We also found that SPL genes control meristem size by repressing WUSCHEL expression via a novel genetic pathway.