Root development is crucial for plant growth and therefore a key factor in plant performance and food production. Arabidopsis thaliana is the most commonly used system to study root system architecture (RSA). Growing plants on agar-based media has always been routine practice, but this approach poorly reflects the natural situation, which fact in recent years has led to a dramatic shift toward studying RSA in soil. Here, we directly compare RSA responses to agar-based medium (plates) and potting soil (rhizotrons) for a set of redundant loss-of-function plethora (plt) CRISPR mutants with variable degrees of secondary root defects. We demonstrate that plt3plt7 and plt3plt5plt7 plants, which produce only a handful of emerged secondary roots, can be distinguished from other genotypes based on both RSA shape and individual traits on plates and rhizotrons. However, in rhizotrons the secondary root density and the total contribution of the side root system to the RSA is increased in these two mutants, effectively rendering their phenotypes less distinct compared to WT. On the other hand, plt3, plt3plt5, and plt5plt7 mutants showed an opposite effect by having reduced secondary root density in rhizotrons. This leads us to believe that plate versus rhizotron responses are genotype dependent, and these differential responses were also observed in unrelated mutants short-root and scarecrow. Our study demonstrates that the type of growth system affects the RSA differently across genotypes, hence the optimal choice of growth conditions to analyze RSA phenotype is not predetermined.

Background: The juxtaposition of newly formed primordia in the root and shoot differs greatly, but their formation in both contexts depends on local accumulation of the signaling molecule auxin. Whether the spacing of lateral roots along the main root and the arrangement of leaf primordia at the plant apex are controlled by related underlying mechanisms has remained unclear.Results: Here, we show that, in Arabidopsis thaliana, three transcriptional regulators implicated in phyllotaxis, PLETHORA3 (PLT3), PLT5, and PLT7, are expressed in incipient lateral root primordia where they are required for primordium development and lateral root emergence. Furthermore, all three PLT proteins prevent the formation of primordia close to one another, because, in their absence, successive lateral root primordia are frequently grouped in close longitudinal or radial clusters. The triple plt mutant phenotype is rescued by PLT-vYFP fusion proteins, which are expressed in the shoot meristem as well as the root, but not by expression of PLT7 in the shoot alone. Expression of all three PLT genes requires auxin response factors ARF7 and ARF19, and the reintroduction of PLT activity suffices to rescue lateral root formation in arf7,arf19.Conclusions: Intriguingly PLT 3, PLT5, and PLT7 not only control the positioning of organs at the shoot meristem but also in the root; a striking observation that raises many evolutionary questions.

The hormone cytokinin (CK) controls root length in Arabidopsis thaliana by defining where dividing cells, derived from stem cells of the root meristem, start to differentiate [1-6]. However, the regulatory inputs directing CK to promote differentiation remain poorly understood. Here, we show that the HD-ZIPIII transcription factor PHABULOSA (PHB) directly activates the CK biosynthesis gene ISOPENTENYL TRANSFERASE 7 (IPT7), thus promoting cell differentiation and regulating root length. We further demonstrate that CK feeds back to repress both PHB and microRNA165, a negative regulator of PHB. These interactions comprise an incoherent regulatory loop in which CK represses both its activator and a repressor of its activator. We propose that this regulatory circuit determines the balance of cell division and differentiation during root development and may provide robustness against CK fluctuations.

Raffaele Dello Ioio,1 Carla Galinha,1 Alexander G. Fletcher,2 Stephen P. Grigg,1,7 Attila Molnar,3 Viola Willemsen,4 Ben Scheres,4 Sabrina Sabatini,5 David Baulcombe,3 Philip K. Maini,2 and Miltos Tsiantis1,6,* 1Department of Plant Sciences, University of Oxford, Oxford OX1 3RB, UK 2Centre for Mathematical Biology, Mathematical Institute, University of Oxford, Oxford OX1 3LB, UK 3Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, UK 4Molecular Genetics, Department of Biology, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands 5Laboratory of Functional Genomics and Proteomics of Model Systems, Dipartimento di Biologia e Biotecnologie, Sapienza Università di Roma, Via dei Sardi 70, 00185 Rome, Italy 6Max Planck Institute for Plant Breeding Research, Carl-von-Linne-Weg 10, D-50829 Cologne, Germany

Lateral organ distribution at the shoot apical meristem defines specific and robust phyllotaxis patterns that have intrigued biologists and mathematicians for centuries. In silico studies have revealed that this self-organizing process can be recapitulated by modeling the polar transport of the phytohormone auxin. Phyllotactic patterns change between species and developmental stages, but the processes behind these variations have remained unknown. Here we use regional complementation experiments to reveal that phyllotactic switches in Arabidopsis shoots can be mediated by PLETHORA-dependent control of local auxin biosynthesis.

The pattern of plant organ initiation at the shoot apical meristem (SAM), termed phyllotaxis, displays regularities that have long intrigued botanists and mathematicians alike. In the SAM, the central zone (CZ) contains a population of stem cells that replenish the surrounding peripheral zone (PZ), where organs are generated in regular patterns. These patterns differ between species and may change in response to developmental or environmental cues [1]. Expression analysis of auxin efflux facilitators of the PIN-FORMED (PIN) family combined with modeling of auxin transport has indicated that organ initiation is associated with intracellular polarization of PIN proteins and auxin accumulation [2-10]. However, regulators that modulate PIN activity to determine phyllotactic patterns have hitherto been unknown. Here we reveal that three redundantly acting PLETHORA (PLT)-like AP2 domain transcription factors control shoot organ positioning in the model plant Arabidopsis thaliana. Loss of PLT3, PLT5, and PLT7 function leads to nonrandom, metastable changes in phyllotaxis. Phyllotactic changes in plt3plt5plt7 mutants are largely attributable to misregulation of PIN1 and can be recapitulated by reducing PIN1 dosage, revealing that PLT proteins are key regulators of PIN1 activity in control of phyllotaxis.

CUP-SHAPED COTYLEDON2 (CUC2) and the interacting microRNA miR164 regulate leaf margin dissection. Here, we further investigate the evolution and the specific roles of the CUC1 to CUC3 genes during Arabidopsis thaliana leaf serration. We show that CUC2 is essential for dissecting the leaves of a wide range of lobed/serrated Arabidopsis lines. Inactivation of CUC3 leads to a partial suppression of the serrations, indicating a role for this gene in leaf shaping. Morphometric analysis of leaf development and genetic analysis provide evidence for different temporal contributions of CUC2 and CUC3. Chimeric constructs mixing CUC regulatory sequences with different coding sequences reveal both redundant and specific roles for the three CUC genes that could be traced back to changes in their expression pattern or protein activity. In particular, we show that CUC1 triggers the formation of leaflets when ectopically expressed instead of CUC2 in the developing leaves. These divergent fates of the CUC1 and CUC2 genes after their formation by the duplication of a common ancestor is consistent with the signature of positive selection detected on the ancestral branch to CUC1. Combining experimental observations with the retraced origin of the CUC genes in the Brassicales, we propose an evolutionary scenario for the CUC genes.

Development of seed plant embryos is polarized along the apical-basal axis. This polarization occurs in the absence of cell migration and culminates in the establishment of two distinct pluripotent cell populations: the shoot apical meristem (SAM) and root meristem (RM), which postembryonically give rise to the entire shoot and root systems of the plant. The acquisition of genetic pathways that delimit root from shoot during embryogenesis must have played a pivotal role during land plant evolution because roots evolved after shoots in ancestral vascular plants and may be shoot-derived organs [1]. However, such pathways are very poorly understood. Here we show that RM establishment in the model plant Arabidopsis thaliana requires apical confinement of the Class III HOMEODOMAIN-LEUCINE ZIPPER (HD-ZIP III) proteins PHABULOSA (PHB) and PHAVOLUTA (PHV), which direct both SAM development and shoot lateral organ polarity [2, 3]. Failure to restrict PHB and PHV-expression apically via a microRNA-dependent pathway prevents correct elaboration of the embryonic root development program and results in embryo lethality. As such, repression of a fundamental shoot development pathway is essential for correct root development. Additionally, our data suggest that a single patterning process, based on HD-ZIP III repression, mediates both apical-basal and radial polarity in the embryo and lateral organ polarity in the shoot.

In the past two years novel connections were described between auxin function and transcription factor patterning systems involved in both leaf initiation and elaboration of leaf axial patterning. A cascade of small RNA-based regulatory steps was suggested to facilitate delimitation of cell types comprising the upper versus lower parts of the leaf. Developmental regulation of cellular growth emerged as a crucial component in regulation of leaf form with TCP and CUC2 transcription factors playing a key role in this process. Finally, cis-regulatory evolution of developmental genes emerged as a process that likely contributed to diversification of leaf form, while studies in seedless land plants have begun to elucidate the ancestral and derived aspects of leaf development pathways.

Plant development is characterized by precise control of gene regulation,leading to the correct spatial and temporal tissue patterning. We have characterized the Arabidopsis jabba-1D (jba-1D) mutant,which displays multiple enlarged shoot meristems, radialized leaves, reduced gynoecia and vascular defects. The jba-1D meristem phenotypes require WUSCHEL (WUS) activity, and correlate with a dramatic increase in WUS expression levels. We demonstrate that the jba-1D phenotypes are caused by over-expression of miR166g,and require the activity of the RNase III helicase DCL1. miR166gover-expression in jba-1D plants affects the transcripts of several class III homeodomain-leucine zipper (AtHD-ZIP) family target genes. The expression of PHABULOSA (PHB), PHAVOLUTA(PHV) and CORONA (CNA) is significantly reduced in a jba-1D background, while REVOLUTA (REV)expression is elevated and ATHB8 is unchanged. In addition, we show that miR166 has a dynamic expression pattern in wild-type and jba-1D embryos. Our analysis demonstrates an indirect role for miRNAs in controlling meristem formation via regulation of WUSexpression, and reveals complex regulation of the class III AtHD-ZIPgene family.

Leaves of flowering plants are determinate organs produced by pluripotent structures termed shoot apical meristems. Once specified, leaves differentiate an adaxial (upper) side specialized for light capture, and an abaxial (lower) side specialized for gas exchange. A functional relationship between meristem activity and the differentiation of adaxial leaf fate has been recognized for over fifty years, but the molecular basis of this interaction is unclear. In Arabidopsis thaliana , activity of the class I KNOX ( KNOTTED1 -like homeobox) genes SHOOTMERISTEMLESS ( STM ) and BREVIPEDICELLUS ( BP ) is required for meristem function but excluded from leaves 1 , 2 , 3 , whereas members of the HD-Zip III (class III homeodomain leucine zipper) protein family function to promote both meristem activity and adaxial leaf fate 4 , 5 , 6 . Here we show that the zinc-finger protein SERRATE acts in a microRNA (miRNA) gene-silencing pathway to regulate expression of the HD-Zip III gene PHABULOSA (PHB) while also limiting the competence of shoot tissue to respond to KNOX expression. Thus, SERRATE acts to coordinately regulate meristem activity and leaf axial patterning.

Eukaryotic development requires the precise spatial and temporal expression of regulatory genes that pattern tissues and control cell fate. It is becoming increasingly clear that small RNA molecules are important participants in these processes, providing sequence specificity for targeted regulation of key developmental factors at the post-transcriptional level. microRNAs (miRNAs) are 21-24 nucleotide single-stranded RNA molecules found in both plants and animals that can down-regulate gene expression by pairing to the messages of protein-coding genes (Bartel, 2004). They are produced from larger precursor transcripts of ~70-300 nucleotides in length that contain a characteristic hairpin secondary structure. In plants, mature miRNA sequences are processed from the double-stranded hairpin region of the precursor molecules by DICER-LIKE1 (Schauer et al., 2002), a ribonuclease III helicase protein that acts as a dsRNA-specific endonuclease (Park et al., 2002; Reinhart et al., 2002). The novel protein HEN1 and the nuclear dsRNA-binding protein HYL1 are also required for plant miRNA accumulation (Han et al., 2004; Park et al., 2002; Vazquez et al., 2004). miRNAs appear to regulate gene expression by binding to complementary sequences in the mRNA transcripts produced by their target genes (Bartel, 2004). While animal miRNAs tend to have several mismatches with their target mRNA sequences, plant miRNAs are characterized by their nearperfect complementarity with their targets (Rhoades et al., 2002). Interaction of the miRNA with its target mRNA sequence is recognized by an RNA-induced silencing complex (RISC), leading either to specific cleavage of the mRNA via RISC endonuclease activity, or to translational repression (reviewed by Bartel, 2004). The RISC has been purified from fly and human cells (Hammond et al., 2000; Martinez et al., 2002), and in both cases contains a member of the ARGONAUTE (AGO) family of PPD proteins (Cerutti et al., 2000). Arabidopsis plants with reduced AGO1 gene activity accumulate mRNAs that are normally targeted for miRNAmediated cleavage, confirming a role for AGO1 in the miRNA regulatory pathway (Vaucheret et al., 2004). Mutants that lack DCL1 activity are embryo lethal (Schauer et al., 2002), revealing that miRNA metabolism is essential for normal plant development. Similarly, plants carrying weaker dcl1 alleles survive embryogenesis but display a wide spectrum of developmental defects (Schauer et al., 2002), while hen1 and hyl1 null mutants show reduced miRNA levels and morphological phenotypes that overlap with those of weak dcl1 alleles (Han et al., 2004; Park et al., 2002; Vazquez et al., 2004). ago1 null mutants are viable but have pleiotropic developmental phenotypes (Bohmert et al., 1998), while ago1 hypomorphic mutants exhibit morphological defects similar to those of dcl1, hen1 and hyl1 mutants (Vaucheret et al., 2004). Consistent with the demonstration that activity of the miRNA pathway is important for plant development, it has been observed that a large fraction of the predicted target transcripts of plant miRNAs encode members of transcription Plant development is characterized by precise control of gene regulation, leading to the correct spatial and temporal tissue patterning. We have characterized the Arabidopsis jabba-1D (jba-1D) mutant, which displays multiple enlarged shoot meristems, radialized leaves, reduced gynoecia and vascular defects. The jba-1D meristem phenotypes require WUSCHEL (WUS) activity, and correlate with a dramatic increase in WUS expression levels. We demonstrate that the jba-1D phenotypes are caused by over-expression of miR166g, and require the activity of the RNase III helicase DCL1. miR166g overexpression in jba-1D plants affects the transcripts of several class III homeodomain-leucine zipper (AtHD-ZIP) family target genes. The expression of PHABULOSA (PHB), PHAVOLUTA (PHV) and CORONA (CNA) is significantly reduced in a jba-1D background, while REVOLUTA (REV) expression is elevated and ATHB8 is unchanged. In addition, we show that miR166 has a dynamic expression pattern in wild-type and jba-1D embryos. Our analysis demonstrates an indirect role for miRNAs in controlling meristem formation via regulation of WUS expression, and reveals complex regulation of the class III AtHD-ZIP gene family.

Leaves, the plant’s major photosynthetic organs, form through the activity of groups of pluripotent cells, termed shoot apical meristems (SAMs), located at the growing tips of plants. Leaves develop with a dorso–ventral asymmetry, with the adaxial surface adjacent to the meristem and the abaxial surface developing at a distance from it. Molecular genetic studies have shown that the correct specification of adaxial/abaxial polarity requires communication between the incipient leaf and the meristem, and that the juxtaposition of adaxial/abaxial fates is necessary for lamina outgrowth (Waites and Hudson 1995; McConnell et al. 2001). Over the last few years, a number of factors that control cell fate specification in the apex have been identified. This review will focus on recent advances on distinct but overlapping aspects of leaf development, namely, the transition from meristem to leaf fate and the specification of abaxial/adaxial polarity and its possible role in leaf growth.