In plants, the phloem distributes photosynthetic products for metabolism and storage over long distances. It relies on specialized cells, the sieve elements, which are enucleated and interconnected through large so-called sieve pores in their adjoining cell walls. Reverse genetics identified PECTATE LYASE-LIKE 12 (PLL12) as critical for plant growth and development. Using genetic complementations, we established that PLL12 is required exclusively late during sieve element differentiation. Structural homology modeling, enzyme inactivation, and overexpression suggest a vital role for PLL12 in sieve-element-specific pectin remodeling. While short distance symplastic diffusion is unaffected, the pll12 mutant is unable to accommodate sustained plant development due to an incapacity to accommodate increasing hydraulic demands on phloem long-distance transport as the plant grows—a defect that is aggravated when combined with another sieve-element-specific mutant callose synthase 7 (cals7). Establishing CALS7 as a specific sieve pore marker, we investigated the subcellular dynamics of callose deposition in the developing sieve plate. Using fluorescent CALS7 then allowed identifying structural defects in pll12 sieve pores that are moderate at the cellular level but become physiologically relevant due to the serial arrangement of sieve elements in the sieve tube. Overall, pectin degradation through PLL12 appears subtle in quantitative terms. We therefore speculate that PLL12 may act as a regulator to locally remove homogalacturonan, thus potentially enabling further extracellular enzymes to access and modify the cell wall during sieve pore maturation.

Plant biomass plays an increasingly important role in the circular bioeconomy, replacing non-renewable fossil resources. Genetic engineering of this lignocellulosic biomass could benefit biorefinery transformation chains by lowering economic and technological barriers to industrial processing. However, previous efforts have mostly targeted the major constituents of woody biomass: cellulose, hemicellulose and lignin. Here we report the engineering of wood structure through the introduction of callose, a polysaccharide novel to most secondary cell walls. Our multiscale analysis of genetically engineered poplar trees shows that callose deposition modulates cell wall porosity, water and lignin contents and increases the lignin–cellulose distance, ultimately resulting in substantially decreased biomass recalcitrance. We provide a model of the wood cell wall nano-architecture engineered to accommodate the hydrated callose inclusions. Ectopic polymer introduction into biomass manifests in new physico-chemical properties and offers new avenues when considering lignocellulose engineering.

Single-cell sequencing has recently allowed the generation of exhaustive root cell atlases. However, some cell types are elusive and remain underrepresented. Here we use a second-generation single-cell approach, where we zoom in on the root transcriptome sorting with specific markers to profile the phloem poles at an unprecedented resolution. Our data highlight the similarities among the developmental trajectories and gene regulatory networks common to protophloem sieve element (PSE)-adjacent lineages in relation to PSE enucleation, a key event in phloem biology. As a signature for early PSE-adjacent lineages, we have identified a set of DNA-binding with one finger (DOF) transcription factors, the PINEAPPLEs (PAPL), that act downstream of PHLOEM EARLY DOF (PEAR) genes and are important to guarantee a proper root nutrition in the transition to autotrophy. Our data provide a holistic view of the phloem poles that act as a functional unit in root development.

Simple mechanisms are sometimes the hardest to prove. For instance, the Münch hypothesis on how phloem tubes move sugars from source leaves to sink tissues remained a just a plausible idea for 86 years until it was experimentally demonstrated in 2016 (Knoblauch et al., 2016). In new work, Mendel Perkins and collaborators (Perkins et al., 2022) tackle another simple long-standing question: how do monolignols, the precursors of lignin, get exported across the plasma membrane to the apoplastic space where lignin polymerization takes place?

In plants, the phloem distributes photosynthetic products for metabolism and storage over long distances. It relies on specialized cells, the sieve elements, which are enucleated and interconnected through large so-called sieve pores in their adjoining cell walls. Reverse genetics identified PECTATE LYASE LIKE 12 ( PLL12 ) as critical for plant growth and development. Using genetic complementation, we establish that PLL12 enzyme activity is required exclusively late during cell differentiation. Physiological assays suggested a role specifically in phloem long distance transport while short distance symplastic transport does not require PLL12 . Exploiting CALLOSE SYNTHASE 7 (CALS7) as a polar fluorescent marker, we identified structural defects in sieve pores in pll12 . Due to the serial arrangement of sieve elements in the phloem, such defects should affect a plant’s ability specifically for symplastic transport over long distances. ### Competing Interest Statement The authors have declared no competing interest.

Single cell sequencing has recently allowed the generation of exhaustive root cell atlases. However, some cell types are elusive and remain underrepresented. Here, we use a second- generation single cell approach, where we zoom in on the root transcriptome sorting with specific markers to profile the phloem poles at an unprecedented resolution. Our data highlight the similarities among the developmental trajectories and gene regulatory networks communal to protophloem sieve element (PSE) adjacent lineages in relation to PSE enucleation, a key event in phloem biology. As a signature for early PSE-adjacent lineages, we have identified a set of DNA-binding with one finger (DOF) transcription factors, the PINEAPPLEs (PAPL), that act downstream of PHLOEM EARLY DOF ( PEAR ) genes, and are important to guarantee a proper root nutrition in the transition to autotrophy. Our data provide a holistic view of the phloem poles that act as a functional unit in root development. ### Competing Interest Statement The authors have declared no competing interest.

Apical growth in plants initiates upon seed germination, whereas radial growth is primed only during early ontogenesis in procambium cells and activated later by the vascular cambium(1). Although it is not known how radial growth is organized and regulated in plants, this system resembles the developmental competence observed in some animal systems, in which pre-existing patterns of developmental potential are established early on(2,3). Here we show that in Arabidopsis the initiation of radial growth occurs around early protophloem-sieve-element cell files of the root procambial tissue. In this domain, cytokinin signalling promotes the expression of a pair of mobile transcription factors-PHLOEM EARLY DOF 1 (PEAR1) and PHLOEM EARLY DOF 2 (PEAR2)-and their four homologues (DOF6, TMO6, OBP2 and HCA2), which we collectively name PEAR proteins. The PEAR proteins form a short-range concentration gradient that peaks at protophloem sieve elements, and activates gene expression that promotes radial growth. The expression and function of PEAR proteins are antagonized by the HD-ZIP III proteins, well-known polarity transcription factors(4)-the expression of which is concentrated in the more-internal domain of radially non-dividing procambial cells by the function of auxin, and mobile miR165 and miR166 microRNAs. The PEAR proteins locally promote transcription of their inhibitory HD-ZIP III genes, and thereby establish a negative-feedback loop that forms a robust boundary that demarks the zone of cell division. Taken together, our data establish that during root procambial development there exists a network in which a module that links PEAR and HD-ZIP III transcription factors integrates spatial information of the hormonal domains and miRNA gradients to provide adjacent zones of dividing and more-quiescent cells, which forms a foundation for further radial growth.

AUTHORS: Shunsuke Miyashima2,3*, Pawel Roszak1,2*, Iris Sevilem2*, Koichi Toyokura1,7**, Bernhard Blob1**, Jung-ok Heo1,2, Nathan Mellor8, Hanna Help-Rinta-Rahko2, Sofia Otero1, Wouter Smet4,5,6, Mark Boekschoten6, Guido Hooiveld6, Kayo Hashimoto3,9, Eva-Sophie Wallner12, Yuki Kondo10, Charles Melnyk11, Thomas Greb12, Keiji Nakajima3, Ross Sozzani13, Anthony Bishopp8, Bert De Rybel4, 5, 6 #, Ykä Helariutta1, 2 #

In plants, a complex mixture of solutes and macromolecules is transported by the phloem. Here, we examined how solutes and macromolecules are separated when they exit the phloem during the unloading process. We used a combination of approaches (non-invasive imaging, 3D-electron microscopy, and mathematical modelling) to show that phloem unloading of solutes in Arabidopsis roots occurs through plasmodesmata by a combination of mass flow and diffusion (convective phloem unloading). During unloading, solutes and proteins are diverted into the phloem-pole pericycle, a tissue connected to the protophloem by a unique class of ‘funnel plasmodesmata’. While solutes are unloaded without restriction, large proteins are released through funnel plasmodesmata in discrete pulses, a phenomenon we refer to as ‘batch unloading’. Unlike solutes, these proteins remain restricted to the phloem-pole pericycle. Our data demonstrate a major role for the phloem-pole pericycle in regulating phloem unloading in roots. unloading. This model suggests that sugars and other small molecules move freely through the funnel plasmodesmata, but large proteins pass through these pores in pulses. Future challenges include finding out exactly how plants control phloem unloading and to investigate whether it is possible to modify the delivery of specific molecules to particular parts of the plant. 1994 Knoblauch 2015 ). We combined a detailed ultrastructural analysis of the cellular interfaces in the phloem unloading zone with the kinetics of phloem unloading of fluorescent solutes and macromolecules obtained by real-time imaging of growing roots. This analysis allowed us to derive new quantitative data on the factors that regulate phloem unloading in Arabidopsis. We report on the presence of a unique class of ‘funnel plas-modesmata’ that are involved specifically in the unloading of molecules into the phloem-pole pericycle. We show, by mathematical modelling, that phloem unloading of small solutes from PSEs is convective, i.e. it occurs continuously by a combination of mass flow and diffusion. In contrast, we find that macromolecules are unloaded in discrete pulses, a phenomenon we refer to as ‘batch unloading’. These macromolecules are diverted specifically into the two phloem-pole pericycle cells that abut each PSE where they are filtered out from the unloaded solutes.

In plants, a complex mixture of solutes and macromolecules is transported by the phloem. Here, we examined how solutes and macromolecules are separated when they exit the phloem during the unloading process. We used a combination of approaches (non-invasive imaging, 3D-electron microscopy, and mathematical modelling) to show that phloem unloading of solutes in Arabidopsis roots occurs through plasmodesmata by a combination of mass flow and diffusion (convective phloem unloading). During unloading, solutes and proteins are diverted into the phloem-pole pericycle, a tissue connected to the protophloem by a unique class of ‘funnel plasmodesmata’. While solutes are unloaded without restriction, large proteins are released through funnel plasmodesmata in discrete pulses, a phenomenon we refer to as ‘batch unloading’. Unlike solutes, these proteins remain restricted to the phloem-pole pericycle. Our data demonstrate a major role for the phloem-pole pericycle in regulating phloem unloading in roots.

Vascular plants have developed highly specialized cells to transport nutrients and developmental signals. The differentiation process includes the degradation of multiple organelles of the sieve element cells (SEs) to facilitate transport and, as a consequence, SEs become dependent on neighboring companion cells (CCs). Despite its importance for phloem function and flowering time control, CCs are still a mysterious cell type. In this review, we gather all the genes known to be expressed in CCs, in different organs and organisms, with the objective of better understanding CC identity and function.

Communication between cells is a crucial step to coordinate organ formation and tissue patterning. In plants, the intercellular transport of metabolites and signalling molecules occur symplastically through membranous structures (named plasmodesmata) that traverse the cell wall to connect the cytoplasm and endoplasmic reticulum of neighbouring cells. This review aims to highlight the importance of symplastic communication in plant development. We revisit current literature reporting the effects of changing plasmodesmata in cell morphogenesis, organ initiation and meristem maintenance and comment on recent work involving the identification of novel plasmodesmata regulators and of mobile developmental proteins and RNA molecules. New opportunities for unravelling the dynamic regulation and function of plasmodesmata are also discussed.

A coordinated transition from cell proliferation to differentiation is crucial for organogenesis. We found that extensive chromatin reorganization, shown here for histone H3 proteins, characterizes cell population dynamics in the root developmental compartments. The canonical H3.1 protein, incorporated during S-phase, is maintained at high levels in cells dividing at a high rate but is massively evicted in cells undergoing their last cell cycle before exit to differentiation. A similar pattern was observed in the quadruple mutant for the H3.1-encoding genes HTR1, HTR2, HTR3, and HTR9 (htr1,2,3,9), in which H3.1 is expressed only from the HTR13 gene. H3 eviction is a fast process occurring within the G2 phase of the last cell cycle, which is longer than G2 in earlier cell cycles. This longer G2 likely contributes to lower the H3.1/H3.3 ratio in cells leaving the root meristem. The high H3.1/H3.3 ratio and H3.1 eviction process also occurs in endocycling cells before differentiation, revealing a common principle of H3 eviction in the proliferating and endocycling domains of the root apex. Mutants in the H3.1 chaperone CAF-1 (fas1-4) maintain a pattern similar to that of wild-type roots. Our studies reveal that H3 incorporation and eviction dynamics identify cells with different cell division potential during organ patterning.

Genome replication in multicellular organisms involves duplication of both the genetic material and the epigenetic information stored in DNA and histones. In some cases, the DNA replication process provides a window of opportunity for resetting chromatin marks in the genome of the future daughter cells instead of transferring them identical copies. This crucial step of genome replication depends on the correct function of DNA replication factors and the coordination between replication and transcription in proliferating cells. In fact, the histone composition and modification status appears to be intimately associated with the proliferation potential of cells within developing organs. Here we discuss these topics in the light of recent advances in our understanding of how genome replication, transcriptional silencing and chromatin dynamics are coordinated in proliferating cells.

Chromatin is a macromolecular complex where DNA associates with histone proteins and a variety of non-histone proteins. Among the 4 histone types present in nucleosomes, histone H3 is encoded by a large number of genes in most eukaryotic species and is the histone that contains the largest variety of potential post-translational modifications in the N-terminal amino acid residues. In addition to centromeric histone H3, 2 major types of histone H3 exist, namely the canonical H3.1 and the variant H3.3. In this article, we review the most recent observations on the distinctive features of plant H3 proteins in terms of their expression and dynamics during the cell cycle and at various developmental stages. We also include a discussion on the histone H3 chaperones that actively participate in H3 deposition, in particular CAF-1, HIRA and ASF1, and on the putative plant homologs of human ATRX and DEK chaperones. Accumulating evidence confirms that the balanced deposition of H3.1 and H3.3 is of primary relevance for cell differentiation during plant organogenesis.