GIGANTEA accelerates wheat heading time through gene interactions converging on FLOWERING LOCUS T1.

Precise regulation of flowering time is critical for cereal crops to synchronize reproductive development with optimum environmental conditions, thereby maximizing grain yield. The plant-specific gene GIGANTEA (GI) plays an important role in the control of flowering time, with additional functions on the circadian clock and plant stress responses. In this study, we show that GI loss-of-function mutants in a photoperiod-sensitive tetraploid wheat background exhibit significant delays in heading time under both long-day (LD) and short-day photoperiods, with stronger effects under LD. However, this interaction between GI and photoperiod is no longer observed in isogenic lines carrying either a photoperiod-insensitive allele in the PHOTOPERIOD1 (PPD1) gene or a loss-of-function allele in EARLY FLOWERING 3 (ELF3), a known repressor of PPD1. These results suggest that the normal circadian regulation of PPD1 is required for the differential effect of GI on heading time in different photoperiods. Using crosses between mutant or transgenic plants of GI and those of critical genes in the flowering regulation pathway, we show that GI accelerates wheat heading time by promoting FLOWERING LOCUS T1 (FT1) expression via interactions with ELF3, VERNALIZATION 2 (VRN2), CONSTANS (CO), and the age-dependent microRNA172-APETALA2 (AP2) pathway, at both transcriptional and protein levels. Our study reveals conserved GI mechanisms between wheat and Arabidopsis but also identifies specific interactions of GI with the distinctive photoperiod and vernalization pathways of the temperate grasses. These results provide valuable knowledge for modulating wheat heading time and engineering new varieties better adapted to a changing environment.

Sequencing 4.3 million mutations in wheat promoters to understand and modify gene expression.

Wheat is an important contributor to global food security, and further improvements are required to feed a growing human population. Functional genetics and genomics tools can help us to understand the function of different genes and to engineer beneficial changes. In this study, we used a promoter capture assay to sequence 2-kb regions upstream of all high-confidence annotated genes from 1,513 mutagenized plants from the tetraploid wheat variety Kronos. We identified 4.3 million induced mutations with an accuracy of 99.8%, resulting in a mutation density of 41.9 mutations per kb. We also remapped Kronos exome capture reads to Chinese Spring RefSeq v1.1, identified 4.7 million mutations, and predicted their effects on annotated genes. Using these predictions, we identified 59% more nonsynonymous substitutions and 49% more truncation mutations than in the original study. To show the biological value of the promoter dataset, we selected two mutations within the promoter of the VRN-A1 vernalization gene. Both mutations, located within transcription factor binding sites, significantly altered VRN-A1 expression, and one reduced the number of spikelets per spike. These publicly available sequenced mutant datasets provide rapid and inexpensive access to induced variation in the promoters and coding regions of most wheat genes. These mutations can be used to understand and modulate gene expression and phenotypes for both basic and commercial applications, where limited governmental regulations can facilitate deployment. These mutant collections, together with gene editing, provide valuable tools to accelerate functional genetic studies in this economically important crop.

An efficient transformation method for genome editing of elite bread wheat cultivars.

An efficient genetic transformation protocol is necessary to edit genes for trait improvement directly in elite bread wheat cultivars. We used a protein fusion between a wheat growth-regulating factor 4 (GRF4) and its interacting factor (GIF1) to develop a reproducible genetic transformation and regeneration protocol, which we then used to successfully transform elite bread wheat cultivars Baj, Kachu, Morocco, Reedling, RL6077, and Sujata in addition to the experimental cultivar Fielder. Immature embryos were transformed with the vector using particle bombardment method. Transformation frequency increased nearly 60-fold with the GRF4-GIF1-containing vectors as compared to the control vector and ranged from ~5% in the cultivar Kachu to 13% in the cultivar RL6077. We then edited two genes that confer resistance against leaf rust and powdery mildew directly in the aforementioned elite cultivars. A wheat promoter, TaU3 or TaU6, to drive the expression of guide RNA was effective in gene editing whereas the OsU3 promoter failed to generate any edits. Editing efficiency was nearly perfect with the wheat promoters. Our protocol has made it possible to edit genes directly in elite wheat cultivars and would be useful for gene editing in other wheat varieties, which have been recalcitrant to transformation thus far.

Optimization of ATAC-seq in wheat seedling roots using INTACT-isolated nuclei.

BACKGROUND: The genetic information contained in the genome of an organism is organized in genes and regulatory elements that control gene expression. The genomes of multiple plants species have already been sequenced and the gene repertory have been annotated, however, cis-regulatory elements remain less characterized, limiting our understanding of genome functionality. These elements act as open platforms for recruiting both positive- and negative-acting transcription factors, and as such, chromatin accessibility is an important signature for their identification. RESULTS: In this work we developed a transgenic INTACT [isolation of nuclei tagged in specific cell types] system in tetraploid wheat for nuclei purifications. Then, we combined the INTACT system together with the assay for transposase-accessible chromatin with sequencing [ATAC-seq] to identify open chromatin regions in wheat root tip samples. Our ATAC-seq results showed a large enrichment of open chromatin regions in intergenic and promoter regions, which is expected for regulatory elements and that is similar to ATAC-seq results obtained in other plant species. In addition, root ATAC-seq peaks showed a significant overlap with a previously published ATAC-seq data from wheat leaf protoplast, indicating a high reproducibility between the two experiments and a large overlap between open chromatin regions in root and leaf tissues. Importantly, we observed overlap between ATAC-seq peaks and cis-regulatory elements that have been functionally validated in wheat, and a good correlation between normalized accessibility and gene expression levels. CONCLUSIONS: We have developed and validated an INTACT system in tetraploid wheat that allows rapid and high-quality nuclei purification from root tips. Those nuclei were successfully used to performed ATAC-seq experiments that revealed open chromatin regions in the wheat genome that will be useful to identify cis-regulatory elements. The INTACT system presented here will facilitate the development of ATAC-seq datasets in other tissues, growth stages, and under different growing conditions to generate a more complete landscape of the accessible DNA regions in the wheat genome.

Interplay among ZF-HD and GRF transcription factors during Arabidopsis leaf development.

The growth-regulating factor (GRF) family of transcriptional factors are involved in the control of leaf size and senescence, inflorescence and root growth, grain size, and plant regeneration. However, there is limited information about the genes regulated by these transcriptional factors, which are in turn responsible for their functions. Using a meta-analysis approach, we identified genes encoding Arabidopsis (Arabidopsis thaliana) zinc-finger homeodomain (ZF-HD) transcriptional factors, as potential targets of the GRFs. We further showed that GRF3 binds to the promoter of one of the members of the ZF-HD family, HOMEOBOX PROTEIN 33 (HB33), and activates its transcription. Increased levels of HB33 led to different modifications in leaf cell number and size that were dependent on its expression levels. Furthermore, we found that expression of HB33 for an extended period during leaf development increased leaf longevity. To cope with the functional redundancy among ZF-HD family members, we generated a dominant repressor version of HB33, HB33-SRDX. Expression of HB33-SRDX from HB33 regulatory regions was seedling-lethal, revealing the importance of the ZF-HD family in plant development. Misexpression of HB33-SRDX in early leaf development caused a reduction in both cell size and number. Interestingly, the loss-of-function of HB33 in lines carrying a GRF3 allele insensitive to miR396 reverted the delay in leaf senescence characteristic of these plants. Our results revealed functions for ZF-HDs in leaf development and linked them to the GRF pathway.

GRF-GIF chimeric proteins enhance in vitro regeneration and Agrobacterium-mediated transformation efficiencies of lettuce (Lactuca spp.).

KEY MESSAGE: GRF-GIF chimeric proteins from multiple source species enhance in vitro regeneration in both wild and cultivated lettuce. In addition, they enhance regeneration in multiple types of lettuce including butterheads, romaines, and crispheads. The ability of plants to regenerate in vitro has been exploited for use in tissue culture systems for plant propagation, plant transformation, and genome editing. The success of in vitro regeneration is often genotype dependent and continues to be a bottleneck for Agrobacterium-mediated transformation and its deployment for improvement of some crop species. Manipulation of transcription factors that play key roles in plant development such as BABY BOOM, WUSCHEL, and GROWTH-REGULATING FACTORs (GRFs) has improved regeneration and transformation efficiencies in several plant species. Here, we compare the efficacy of GRF-GIF gene fusions from multiple species to boost regeneration efficiency and shooting frequency in four genotypes of wild and cultivated lettuce (Lactuca spp. L.). In addition, we show that GRF-GIFs with mutated miRNA 396 binding sites increase regeneration efficiency and shooting frequency when compared to controls. We also present a co-transformation strategy for increased transformation efficiency and recovery of transgenic plants harboring a gene of interest. This strategy will enhance the recovery of transgenic plants of other lettuce genotypes and likely other crops in the Compositae family.

A second-generation capture panel for cost-effective sequencing of genome regulatory regions in wheat and relatives.

As genome resources for wheat (Triticum L.) expand at a rapid pace, it is important to update targeted sequencing tools to incorporate improved sequence assemblies and regions of previously unknown significance. Here, we developed an updated regulatory region enrichment capture for wheat and other Triticeae species. The core target space includes sequences from 2-Kbp upstream of each gene predicted in the Chinese Spring wheat genome (IWGSC RefSeq Annotation v1.0) and regions of open chromatin identified with an assay for transposase-accessible chromatin using sequencing from wheat leaf and root samples. To improve specificity, we aggressively filtered candidate repetitive sequences using a combination of nucleotide basic local alignment search tool (BLASTN) searches to the Triticeae Repetitive Sequence Database (TREP), identification of regions with read over-coverage from previous target enrichment experiments, and k-mer frequency analyses. The final design comprises 216.5 Mbp of predicted hybridization space in hexaploid wheat and showed increased specificity and coverage of targeted sequences relative to previous protocols. Test captures on hexaploid and tetraploid wheat and other diploid cereals show that the assay has broad potential utility for cost-effective promoter and open chromatin resequencing and general-purpose genotyping of various Triticeae species.

Recent advances in crop transformation technologies.

Agriculture is experiencing a technological inflection point in its history, while also facing unprecedented challenges posed by human population growth and global climate changes. Key advancements in precise genome editing and new methods for rapid generation of bioengineered crops promise to both revolutionize the speed and breadth of breeding programmes and increase our ability to feed and sustain human population growth. Although genome editing enables targeted and specific modifications of DNA sequences, several existing barriers prevent the widespread adoption of editing technologies for basic and applied research in established and emerging crop species. Inefficient methods for the transformation and regeneration of recalcitrant species and the genotype dependency of the transformation process remain major hurdles. These limitations are frequent in monocotyledonous crops, which alone provide most of the calories consumed by human populations. Somatic embryogenesis and de novo induction of meristems - pluripotent groups of stem cells responsible for plant developmental plasticity - are essential strategies to quickly generate transformed plants. Here we review recent discoveries that are rapidly advancing nuclear transformation technologies and promise to overcome the obstacles that have so far impeded the widespread adoption of genome editing in crop species.

MiR172-APETALA2-like genes integrate vernalization and plant age to control flowering time in wheat.

Plants possess regulatory mechanisms that allow them to flower under conditions that maximize reproductive success. Selection of natural variants affecting those mechanisms has been critical in agriculture to modulate the flowering response of crops to specific environments and to increase yield. In the temperate cereals, wheat and barley, the photoperiod and vernalization pathways explain most of the natural variation in flowering time. However, other pathways also participate in fine-tuning the flowering response. In this work, we integrate the conserved microRNA miR172 and its targets APETALA2-like (AP2L) genes into the temperate grass flowering network involving VERNALIZATION 1 (VRN1), VRN2 and FLOWERING LOCUS T 1 (FT1 = VRN3) genes. Using mutants, transgenics and different growing conditions, we show that miR172 promotes flowering in wheat, while its target genes AP2L1 (TaTOE1) and AP2L5 (Q) act as flowering repressors. Moreover, we reveal that the miR172-AP2L pathway regulates FT1 expression in the leaves, and that this regulation is independent of VRN2 and VRN1. In addition, we show that the miR172-AP2L module and flowering are both controlled by plant age through miR156 in spring cultivars. However, in winter cultivars, flowering and the regulation of AP2L1 expression are decoupled from miR156 downregulation with age, and induction of VRN1 by vernalization is required to repress AP2L1 in the leaves and promote flowering. Interestingly, the levels of miR172 and both AP2L genes modulate the flowering response to different vernalization treatments in winter cultivars. In summary, our results show that conserved and grass specific gene networks interact to modulate the flowering response, and that natural or induced mutations in AP2L genes are useful tools for fine-tuning wheat flowering time in a changing environment.

Interactions between SQUAMOSA and SHORT VEGETATIVE PHASE MADS-box proteins regulate meristem transitions during wheat spike development.

Inflorescence architecture is an important determinant of crop productivity. The number of spikelets produced by the wheat inflorescence meristem (IM) before its transition to a terminal spikelet (TS) influences the maximum number of grains per spike. Wheat MADS-box genes VERNALIZATION 1 (VRN1) and FRUITFULL 2 (FUL2) (in the SQUAMOSA-clade) are essential to promote the transition from IM to TS and for spikelet development. Here we show that SQUAMOSA genes contribute to spikelet identity by repressing MADS-box genes VEGETATIVE TO REPRODUCTIVE TRANSITION 2 (VRT2), SHORT VEGETATIVE PHASE 1 (SVP1), and SVP3 in the SVP clade. Constitutive expression of VRT2 resulted in leafy glumes and lemmas, reversion of spikelets to spikes, and downregulation of MADS-box genes involved in floret development, whereas the vrt2 mutant reduced vegetative characteristics in spikelets of squamosa mutants. Interestingly, the vrt2 svp1 mutant showed similar phenotypes to squamosa mutants regarding heading time, plant height, and spikelets per spike, but it exhibited unusual axillary inflorescences in the elongating stem. We propose that SQUAMOSA-SVP interactions are important to promote heading, formation of the TS, and stem elongation during the early reproductive phase, and that downregulation of SVP genes is then necessary for normal spikelet and floral development. Manipulating SVP and SQUAMOSA genes can contribute to engineering spike architectures with improved productivity.

ARF2 represses expression of plant GRF transcription factors in a complementary mechanism to microRNA miR396.

Members of the GROWTH REGULATING FACTOR (GRF) family of transcription factors play key roles in the promotion of plant growth and development. Many GRFs are post-transcriptionally repressed by microRNA (miRNA) miR396, an evolutionarily conserved small RNA, which restricts their expression to proliferative tissue. We performed a comprehensive analysis of the GRF family in eudicot plants and found that in many species all the GRFs have a miR396-binding site. Yet, we also identified GRFs with mutations in the sequence recognized by miR396, suggesting a partial or complete release of their post-transcriptional repression. Interestingly, Brassicaceae species share a group of GRFs that lack miR396 regulation, including Arabidopsis GRF5 and GRF6. We show that instead of miR396-mediated post-transcriptional regulation, the spatiotemporal control of GRF5 is achieved through evolutionarily conserved promoter sequences, and that AUXIN RESPONSE FACTOR 2 (ARF2) binds to such conserved sequences to repress GRF5 expression. Furthermore, we demonstrate that the unchecked expression of GRF5 in arf2 mutants is responsible for the increased cell number of arf2 leaves. The results describe a switch in the repression mechanisms that control the expression of GRFs and mechanistically link the control of leaf growth by miR396, GRFs, and ARF2 transcription factors.

A GRF-GIF chimeric protein improves the regeneration efficiency of transgenic plants.

The potential of genome editing to improve the agronomic performance of crops is often limited by low plant regeneration efficiencies and few transformable genotypes. Here, we show that expression of a fusion protein combining wheat GROWTH-REGULATING FACTOR 4 (GRF4) and its cofactor GRF-INTERACTING FACTOR 1 (GIF1) substantially increases the efficiency and speed of regeneration in wheat, triticale and rice and increases the number of transformable wheat genotypes. GRF4-GIF1 transgenic plants were fertile and without obvious developmental defects. Moreover, GRF4-GIF1 induced efficient wheat regeneration in the absence of exogenous cytokinins, which facilitates selection of transgenic plants without selectable markers. We also combined GRF4-GIF1 with CRISPR-Cas9 genome editing and generated 30 edited wheat plants with disruptions in the gene Q (AP2L-A5). Finally, we show that a dicot GRF-GIF chimera improves regeneration efficiency in citrus, suggesting that this strategy can be applied to dicot crops.

APETALA 2-like genes AP2L2 and Q specify lemma identity and axillary floral meristem development in wheat.

The spikelet is the basic unit of the grass inflorescence. In tetraploid (Triticum turgidum) and hexaploid wheat (Triticum aestivum), the spikelet is a short indeterminate branch with two proximal sterile bracts (glumes) followed by a variable number of florets, each including a bract (lemma) with an axillary flower. Varying levels of miR172 and/or its target gene Q (AP2L5) result in gradual transitions of glumes to lemmas, and vice versa. Here, we show that AP2L5 and its related paralog AP2L2 play critical and redundant roles in the specification of axillary floral meristems and lemma identity. AP2L2, also targeted by miR172, displayed similar expression profiles to AP2L5 during spikelet development. Loss-of-function mutants in both homeologs of AP2L2 (henceforth ap2l2) developed normal spikelets, but ap2l2 ap2l5 double mutants generated spikelets with multiple empty bracts before transitioning to florets. The coordinated nature of these changes suggest an early role of these genes in floret development. Moreover, the flowers of ap2l2 ap2l5 mutants showed organ defects in paleas and lodicules, including the homeotic conversion of lodicules into carpels. Mutations in the miR172 target site of AP2L2 were associated with reduced plant height, more compact spikes, promotion of lemma-like characters in glumes and smaller lodicules. Taken together, our results show that the balance in the expression of miR172 and AP2-like genes is crucial for the correct development of spikelets and florets, and that this balance has been altered during the process of wheat and barley (Hordeum vulgare) domestication. The manipulation of this regulatory module provides an opportunity to modify spikelet architecture and improve grain yield.

Robust increase of leaf size by Arabidopsis thaliana GRF3-like transcription factors under different growth conditions.

An increase in crop yield is essential to reassure food security to meet the accelerating global demand. Several genetic modifications can increase organ size, which in turn might boost crop yield. Still, only in a few cases their performance has been evaluated under stress conditions. MicroRNA miR396 repress the expression of GROWTH-REGULATING FACTOR (GRF) genes that codes for transcription factors that promote organ growth. Here, we show that both Arabidopsis thaliana At-GRF2 and At-GRF3 genes resistant to miR396 activity (rGRF2 and rGRF3) increased organ size, but only rGRF3 can produce this effect without causing morphological defects. Furthermore, introduction of At-rGRF3 in Brassica oleracea can increase organ size, and when At-rGRF3 homologs from soybean and rice are introduced in Arabidopsis, leaf size is also increased. This suggests that regulation of GRF3 activity by miR396 is important for organ growth in a broad range of species. Plants harboring rGRF3 have larger leaves also under drought stress, a condition that stimulates miR396 accumulation. These plants also showed an increase in the resistance to virulent bacteria, suggesting that the size increment promoted by rGRF3 occurs without an obvious cost on plant defenses. Our findings indicate that rGRF3 can increase plant organ size under both normal and stress conditions and is a valuable tool for biotechnological applications.

GIF Transcriptional Coregulators Control Root Meristem Homeostasis.

In the root meristem, the quiescent center (QC) is surrounded by stem cells, which in turn generate the different cell types of the root. QC cells rarely divide under normal conditions but can replenish damaged stem cells. In the proximal meristem, the daughters of stem cells, which are referred to as transit-amplifying cells, undergo additional rounds of cell division prior to differentiation. Here, we describe the functions of GRF-INTERACTING FACTORs (GIFs), including ANGUSTIFOLIA3 (AN3), in Arabidopsis thaliana roots. GIFs have been shown to interact with GRF transcription factors and SWI/SNF chromatin remodeling complexes. We found that combinations of GIF mutants cause the loss of QC identity. However, despite their QC impairment, GIF mutants have a significantly enlarged root meristem with additional lateral root cap layers. We show that the increased expression of PLETHORA1 (PLT1) is at least partially responsible for the large root meristems of an3 mutants. Furthermore, we found that GIFs are necessary for maintaining the precise expression patterns of key developmental regulators and that AN3 complexes bind directly to the promoter regions of PLT1 as well as SCARECROW We propose that AN3/GIFs participate in different pathways that control QC organization and the size of the meristem.

Evolutionary Footprints Reveal Insights into Plant MicroRNA Biogenesis.

MicroRNAs (miRNAs) are endogenous small RNAs that recognize target sequences by base complementarity and play a role in the regulation of target gene expression. They are processed from longer precursor molecules that harbor a fold-back structure. Plant miRNA precursors are quite variable in size and shape, and are recognized by the processing machinery in different ways. However, ancient miRNAs and their binding sites in target genes are conserved during evolution. Here, we designed a strategy to systematically analyze MIRNAs from different species generating a graphical representation of the conservation of the primary sequence and secondary structure. We found that plant MIRNAs have evolutionary footprints that go beyond the small RNA sequence itself, yet their location along the precursor depends on the specific MIRNA We show that these conserved regions correspond to structural determinants recognized during the biogenesis of plant miRNAs. Furthermore, we found that the members of the miR166 family have unusual conservation patterns and demonstrated that the recognition of these precursors in vivo differs from other known miRNAs. Our results describe a link between the evolutionary conservation of plant MIRNAs and the mechanisms underlying the biogenesis of these small RNAs and show that the MIRNA pattern of conservation can be used to infer the mode of miRNA biogenesis.

microRNA172 plays a crucial role in wheat spike morphogenesis and grain threshability.

Wheat domestication from wild species involved mutations in the Q gene. The q allele (wild wheats) is associated with elongated spikes and hulled grains, whereas the mutant Q allele (domesticated wheats) confers subcompact spikes and free-threshing grains. Previous studies showed that Q encodes an AP2-like transcription factor, but the causal polymorphism of the domestication traits remained unclear. Here, we show that the interaction between microRNA172 (miR172) and the Q allele is reduced by a single nucleotide polymorphism in the miRNA binding site. Inhibition of miR172 activity by a miRNA target mimic resulted in compact spikes and transition from glumes to florets in apical spikelets. By contrast, overexpression of miR172 was sufficient to induce elongated spikes and non-free-threshing grains, similar to those observed in three Q loss-of-function mutations. These lines showed transitions from florets to glumes in the basal spikelets. These localized homeotic changes were associated with opposing miR172/Q gradients along the spike. We propose that the selection of a nucleotide change at the miR172 binding site of Q contributed to subcompact spikes and free-threshing grains during wheat domestication.

RNA-seq studies using wheat PHYTOCHROME B and PHYTOCHROME C mutants reveal shared and specific functions in the regulation of flowering and shade-avoidance pathways.

BACKGROUND: In cereal crops such as wheat, an optimal timing of developmental transitions is required to maximize grain yield. Many of these developmental changes are precisely regulated by changes in the duration, intensity or quality of light. Phytochromes are dimeric photoreceptors that absorb light maximally in the red and far-red wavelengths and induce large-scale transcriptional changes in response to variation in light quality. In wheat, PHYC is required for early flowering under long days. However, it is currently unknown whether this function requires the presence of PHYB. In this study, we characterized the role of PHYB in wheat development and used RNA-seq to analyze and compare the transcriptomes of phyB-null and phyC-null TILLING mutants. RESULTS: Under long-day photoperiods, phyB-null plants exhibit a severe delay in flowering comparable to the delay observed in phyC-null plants. These results demonstrate that both genes are required for the induction of wheat flowering under long days. Using replicated RNA-seq studies we identified 82 genes that are significantly up or down regulated in both the phyB-null and phyC-null mutant relative to their respective wild-type controls. Among these genes are several well-characterized positive regulators of flowering, including PPD1, FT1 and VRN1. Eight-fold more genes were differentially regulated only in the phyB-null mutant (2202) than only in the phyC-null mutant (261). The PHYB-regulated genes were enriched in components of the auxin, gibberellin and brassinosteroid biosynthesis and signaling pathways, and in transcription factors with putative roles in regulating vegetative development and shade-avoidance responses. Several genes involved in abiotic stress tolerance pathways were also found to be regulated by PHYB. CONCLUSIONS: PHYB and PHYC are both required for the photoperiodic induction of wheat flowering, whereas PHYB alone regulates a large number of genes involved in hormone biosynthesis and signaling, shade-avoidance response, and abiotic stress tolerance. Our analysis provides a comprehensive overview of the PHYB- and PHYC-mediated transcriptional changes during light signaling, and an initial step towards the dissection of this regulatory gene network in wheat. This further dissection will be required to explore the individual phytochrome-mediated developmental responses and to evaluate their potential to improve wheat adaptation to changing environments.

Control of cell proliferation and elongation by miR396.

The combinatory effects of cell proliferation and cell elongation determines the rate at which organs growth. In the root meristematic zone cells both divide and expand, while post-mitotic cells in the elongation zone only expands until they reach their final size. The transcription factors of the GROWTH-REGULATING FACTOR (GRF) class promote cell proliferation in various plant organs. Their expression is restricted to cells with a high proliferative capacity, yet strong downregulation of the GRF activity compromise the plant survival. Part of expression pattern of the GRFs is ensured by the post-transcriptional repression mediated by the conserved microRNA miR396. Here we show the quantitative effects in root growth caused by GRF depletion in a series of transgenic lines with different miR396 levels. We show that high miRNA levels affect cell elongation and proliferation in roots. Detailed analysis suggests that cell proliferation is restricted due to a reduction in cell cycle speed that might result from defects in the accumulation of mitotic cyclins. The results provide insights into the participation of the miRNA-GRF regulatory network in root development.