SHORTROOT-Mediated Intercellular Signals Coordinate Phloem Development in Arabidopsis Roots

Asymmetric cell division (ACD) and positional signals play critical roles in the tissue patterning process. In the Arabidopsis (Arabidopsis thaliana) root meristem, two major phloem cell types arise via ACDs of distinct origins: one for companion cells (CCs) and the other for proto- and metaphloem sieve elements (SEs). The molecular mechanisms underlying each of these processes have been reported; however, how these are coordinated has remained elusive. Here, we report a new phloem development process coordinated via the SHORTROOT (SHR) transcription factor in Arabidopsis. The movement of SHR into the endodermis regulates the ACD for CC formation by activating microRNA165/6, while SHR moving into the phloem regulates the ACD generating the two phloem SEs. In the phloem, SHR sequentially activates NAC-REGULATED SEED MORPHOLOGY 1 (NARS1) and SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN 2 (SND2), and these three together form a positive feedforward loop. Under this regulatory scheme, NARS1, generated in the CCs of the root differentiation zone, establishes a top-down signal that drives the ACD for phloem SEs in the meristem. SND2 appears to function downstream to amplify NARS1 via positive feedback. This new regulatory mechanism expands our understanding of the sophisticated vascular tissue patterning processes occurring during postembryonic root development.Peer reviewe

Baseline characteristics of the Korean genetic cohort of inherited cystic kidney disease

Background Identifying genetic mutations in individuals with inherited cystic kidney disease is necessary for precise treatment. We aimed to elucidate the genetic characteristics of cystic kidney disease in the Korean population. Methods We conducted a 3-year prospective, multicenter cohort study at eight hospitals from May 2019 to May 2022. Patients with more than three renal cysts were enrolled and classified into two categories, typical autosomal dominant polycystic kidney disease (ADPKD) and atypical PKD. We identified the clinical characteristics and performed a genetic analysis using a targeted gene panel. Results A total of 725 adult patients were included in the study, of which 560 (77.2%) were diagnosed with typical ADPKD and 165 (22.8%) had atypical PKD. Among the typical ADPKD cases, the Mayo imaging classification was as follows: 1A (55, 9.9%), 1B (149, 26.6%), 1C (198, 35.8%), 1D (90, 16.3%), and 1E (61, 11.0%). The atypical PKD cases were classified as bilateral cystic with bilateral atrophic (31, 37.3%), lopsided (27, 32.5%), unilateral (nine, 10.8%), segmental (eight, 9.6%), bilateral cystic with unilateral atrophic (seven, 8.4%), and asymmetric (one, 1.2%). Pathogenic variants were found in 64.3% of the patients using the ciliopathy-related targeted gene panel. The typical ADPKD group demonstrated a higher discovery rate (62.3%) than the atypical PKD group (41.8%). Conclusion We present a nationwide genetic cohort’s baseline clinical and genetic characteristics for Korean cystic kidney disease

WEREWOLF, a Regulator of Root Hair Pattern Formation, Controls Flowering Time through the Regulation of FT mRNA Stability1[C][W][OA]

A key floral activator, FT, integrates stimuli from long-day, vernalization, and autonomous pathways and triggers flowering by directly regulating floral meristem identity genes in Arabidopsis (Arabidopsis thaliana). Since a small amount of FT transcript is sufficient for flowering, the FT level is strictly regulated by diverse genes. In this study, we show that WEREWOLF (WER), a MYB transcription factor regulating root hair pattern, is another regulator of FT. The mutant wer flowers late in long days but normal in short days and shows a weak sensitivity to vernalization, which indicates that WER controls flowering time through the photoperiod pathway. The expression and double mutant analyses showed that WER modulates FT transcript level independent of CONSTANS and FLOWERING LOCUS C. The histological analysis of WER shows that it is expressed in the epidermis of leaves, where FT is not expressed. Consistently, WER regulates not the transcription but the stability of FT mRNA. Our results reveal a novel regulatory mechanism of FT that is non cell autonomous

PHABULOSA controls the quiescent center-independent root meristem activities in Arabidopsis thaliana.

Plant growth depends on stem cell niches in meristems. In the root apical meristem, the quiescent center (QC) cells form a niche together with the surrounding stem cells. Stem cells produce daughter cells that are displaced into a transit-amplifying (TA) domain of the root meristem. TA cells divide several times to provide cells for growth. SHORTROOT (SHR) and SCARECROW (SCR) are key regulators of the stem cell niche. Cytokinin controls TA cell activities in a dose-dependent manner. Although the regulatory programs in each compartment of the root meristem have been identified, it is still unclear how they coordinate one another. Here, we investigate how PHABULOSA (PHB), under the posttranscriptional control of SHR and SCR, regulates TA cell activities. The root meristem and growth defects in shr or scr mutants were significantly recovered in the shr phb or scr phb double mutant, respectively. This rescue in root growth occurs in the absence of a QC. Conversely, when the modified PHB, which is highly resistant to microRNA, was expressed throughout the stele of the wild-type root meristem, root growth became very similar to that observed in the shr; however, the identity of the QC was unaffected. Interestingly, a moderate increase in PHB resulted in a root meristem phenotype similar to that observed following the application of high levels of cytokinin. Our protoplast assay and transgenic approach using ARR10 suggest that the depletion of TA cells by high PHB in the stele occurs via the repression of B-ARR activities. This regulatory mechanism seems to help to maintain the cytokinin homeostasis in the meristem. Taken together, our study suggests that PHB can dynamically regulate TA cell activities in a QC-independent manner, and that the SHR-PHB pathway enables a robust root growth system by coordinating the stem cell niche and TA domain

PHB suppresses B-ARR activities in the presence of high cytokinin.

<p>A protoplast assay that measures the ARR10 activities in the presence of PHB. <b>(A)</b> A high dosage of PHB (<i>p35S:PHB-em</i> and <i>p35S:PHB</i>) suppressed ARR10 activities under the high cytokinin. For the reporter assay, <i>pUBQ:rLUC</i> was co-transfected as an internal transfection control. Relative TCS activities with effectors (fold induction) were inferred by measuring the ratios between luminescence from fire fly luciferase (<i>pTCS:fLUC</i>) and luminescence from renilla luciferase (<i>pUBQ:rLUC</i>) and then dividing those values with the ratio obtained from control without an effector and BAP treatment. The error bar represents standard deviation. (<b>B</b>) <i>PHB</i> expression from 100 nM BAP-treated protoplasts is analyzed by real-time RT-PCR. Relative <i>PHB</i> expression was obtained by measuring fold changes against <i>PHB</i> expression in the control protoplasts. The error bar represents standard deviation (n = 3). (<b>C</b>) Increasing ARR10 levels by expressing it under <i>WOL</i> promoter restores the root growth in the <i>phb-7D</i>. BAP, 6-benzylaminopurine.</p

The SHR-PHB pathway controls root growth in a cytokinin-dependent manner (A) Relative mRNA levels of <i>IPT3</i> and <i>IPT7</i> in <i>shr, shr phb</i>, and <i>pWOL:PHB-em:GFP</i>_<i>NLS</i> (<i>PHB-em</i>) roots.

<p>Data are normalized to Expression levels in wild-type roots. (<b>B</b>) Root lengths (10 DAG) and (<b>C</b>) meristem images (5 DAG) in <i>shr, shr ipt3-2 ipt7-1</i> mutants. The error bars represent the standard error (n = 20 plants). Scale bar: 50 μm. The white arrowhead marks the end of the meristem.</p

PHB in the stele regulates root meristem and growth activity in a QC-independent manner.

<p>(<b>A)</b> A comparison of root lengths in wild-type, <i>pWOL:PHB-m:GFP_NLS</i>, p<i>WOL:PHB-em:GFP_NLS</i> and <i>shr-2</i> plants (7 DAG). The error bars represent the standard error (n = 9–14 plants). (<b>B, C</b>) Quantitative comparison of PHB-GFP levels in the root stele cells expressing <i>pWOL:PHB-m:GFP</i>_<i>NLS</i> and <i>pWOL:PHB-em:GFP</i>_<i>NLS</i>. Fluorescence intensity was measured for GFP in the boxed area of the panel (<b>B</b>). Error bars represent standard error (n = 11) (<b>D</b>) <i>pCycB1.2:GUS</i> expression shows drastic reduction in cell division potential in the proximal meristem. Expression of <i>pQC25:GUS</i> (<b>E</b>) and <i>pWOX5:YFP</i> (<b>F</b>) in the <i>PHB-em</i> roots. (<b>G</b>) Starch granule accumulation as visualized by Lugol’s staining in the <i>pWOL:PHB-em:GFP</i>_<i>NLS</i> roots. The black arrowheads and red arrows indicate the QC and columella stem cells, respectively. Scale bars: B, 20 μm; D-G, 25 μm.</p

The SHR-PHB pathway and cytokinin signaling.

<p>(<b>A</b>) Cytokinin (CK) expression in wild-type, <i>shr</i>, and <i>shr phb</i> roots. tZ, Transzeatin; tZR, tZ Riboside; iP9G, N⁶-(Δ²-isopentenyl) adenine-9-glucoside; tZ9G, tZ-9-glucoside. (<b>B</b>) Expression of <i>pTCS:GFP</i> in wild-type, <i>shr</i>, and <i>shr phb</i> roots. (C) Real-time PCR and ChIP showing PHB binding to the ARR7 promoter. Line diagram represents the <i>ARR7</i> promoter region. Arrows and blue bars indicate primers and B-ARR binding elements (GGATT/AATCT), respectively. (<b>D</b>) Recovery in <i>pAHP6:erGFP</i> expression in the <i>shr phb</i> roots in comparison with <i>shr</i> roots. The error bars represent the standard deviation (A) and standard error (C) (n = 3 biological replicates). Scale bars: 25 μm. CK, cytokinin.</p

Downstream genes of SHR-PHB in the root stele.

<p>(<b>A</b>) Hierarchical clustering of expression in the <i>Arabidopsis</i> root along the longitudinal axis for genes that are repressed (blue; genes in cluster 2 and 3 in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004973#pgen.1004973.s006" target="_blank">S6C Fig.</a>) or activated (red; genes in cluster 1 in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004973#pgen.1004973.s006" target="_blank">S6C Fig.</a>) by a high level of PHB in <i>shr</i> mutants. Expression values are normalized by row. (<b>B</b>) Over-represented biological functions of genes that are repressed (<i>P</i>-value marked in blue) or (<b>C</b>) activated (<i>P</i>-value marked in red) by a high level of PHB in <i>shr</i> mutants.</p