Antia Rodriguez-Villalon

Phytoene synthase activity controls the biosynthesis of carotenoids and the supply of their metabolic precursors in dark-grown Arabidopsis seedlings

Carotenoids are plastidial isoprenoids essential for plant life. In Arabidopsis thaliana carotenoid biosynthesis is strongly upregulated when seedlings that germinate in the dark (etiolated) emerge from the soil and light derepresses photomorphogenesis, causing etioplasts to become chloroplasts. We found that carotenoid biosynthesis is also induced when deetiolation is derepressed in the absence of actual light, eventually resulting in improved greening (chlorophyll accumulation) upon illumination. The increased production of carotenoids in the dark correlates with an upregulated activity of phytoene synthase (PSY; the first committed enzyme of carotenogenesis) and the induction of PSY gene expression in cotyledons (where carotenoids accumulate in dark-grown seedlings). The metabolic precursors for carotenoid synthesis under these conditions are mostly supplied by the plastidial methylerythritol 4-phosphate (MEP) pathway. Accumulation of flux-controlling MEP pathway enzymes, such as deoxyxylulose 5-phosphate synthase (DXS), is post-transcriptionally increased when deetiolation is derepressed in the dark. Unlike the situation observed in light-grown plants, however, the sole overexpression of DXS in dark-grown seedlings does not increase carotenoid accumulation. By contrast, induced expression of a PSY-encoding transgene results in increased carotenoid levels and a concomitant post-transcriptional accumulation of DXS. These data provide evidence for a feedback mechanism by which PSY controls metabolic flux to the carotenoid pathway in plants.

Suppression of Arabidopsis protophloem differentiation and root meristem growth by CLE45 requires the receptor-like kinase BAM3

Peptide signaling presumably occupies a central role in plant development, yet only few concrete examples of receptor-ligand pairs that act in the context of specific differentiation processes have been described. Here we report that second-site null mutations in the Arabidopsis leucine-rich repeat receptor-like kinase gene barely any meristem 3 (BAM3) perfectly suppress the postembryonic root meristem growth defect and the associated perturbed protophloem development of the brevis radix (brx) mutant. The roots of bam3 mutants specifically resist growth inhibition by the CLAVATA3/ENDOSPERM SURROUNDING REGION 45 (CLE45) peptide ligand. WT plants transformed with a construct for ectopic overexpression of CLE45 could not be recovered, with the exception of a single severely dwarfed and sterile plant that eventually died. By contrast, we obtained numerous transgenic bam3 mutants transformed with the same construct. These transgenic plants displayed a WT phenotype, however, supporting the notion that CLE45 is the likely BAM3 ligand. The results correlate with the observation that external CLE45 application represses protophloem differentiation in WT, but not in bam3 mutants. BAM3, BRX, and CLE45 are expressed in a similar spatiotemporal trend along the developing protophloem, up to the end of the transition zone. Induction of BAM3 expression upon CLE45 application, ectopic overexpression of BAM3 in brx root meristems, and laser ablation experiments suggest that intertwined regulatory activity of BRX, BAM3, and CLE45 could be involved in the proper transition of protophloem cells from proliferation to differentiation, thereby impinging on postembryonic growth capacity of the root meristem.

Molecular genetic framework for protophloem formation

Significance The emergence of vascular tissues played a central role in the plant conquest of land. Both xylem and phloem are essential for the development of flowering plants, yet little is known about the molecular genetic control of phloem specification and differentiation. Here we show that delicate quantitative interplay between two opposing signaling pathways determines cellular commitment to protophloem sieve element fate in root meristems of the model plant Arabidopsis thaliana. Our data suggest that a recently described phloem-specific protein is a positive, quantitative master regulator of phloem fate. The phloem performs essential systemic functions in tracheophytes, yet little is known about its molecular genetic specification. Here we show that application of the peptide ligand CLAVATA3/EMBRYO SURROUNDING REGION 45 (CLE45) specifically inhibits specification of protophloem in Arabidopsis roots by locking the sieve element precursor cell in its preceding developmental state. CLE45 treatment, as well as viable transgenic expression of a weak CLE45G6T variant, interferes not only with commitment to sieve element fate but also with the formative sieve element precursor cell division that creates protophloem and metaphloem cell files. However, the absence of this division appears to be a secondary effect of discontinuous sieve element files and subsequent systemically reduced auxin signaling in the root meristem. In the absence of the formative sieve element precursor cell division, metaphloem identity is seemingly adopted by the normally procambial cell file instead, pointing to possibly independent positional cues for metaphloem formation. The protophloem formation and differentiation defects in brevis radix (brx) and octopus (ops) mutants are similar to those observed in transgenic seedlings with increased CLE45 activity and can be rescued by loss of function of a putative CLE45 receptor, BARELY ANY MERISTEM 3 (BAM3). Conversely, a dominant gain-of-function ops allele or mild OPS dosage increase suppresses brx defects and confers CLE45 resistance. Thus, our data suggest that delicate quantitative interplay between the opposing activities of BAM3-mediated CLE45 signals and OPS-dependent signals determines cellular commitment to protophloem sieve element fate, with OPS acting as a positive, quantitative master regulator of phloem fate.

Primary root protophloem differentiation requires balanced phosphatidylinositol-4,5-biphosphate levels and systemically affects root branching.

Protophloem is a specialized vascular tissue in growing plant organs, such as root meristems. In Arabidopsis mutants with impaired primary root protophloem differentiation, brevis radix (brx) and octopus (ops), meristematic activity and consequently overall root growth are strongly reduced. Second site mutation in the protophloem-specific presumed phosphoinositide 5-phosphatase COTYLEDON VASCULAR PATTERN 2 (CVP2), but not in its homolog CVP2-LIKE 1 (CVL1), partially rescues brx defects. Consistent with this finding, CVP2 hyperactivity in a wild-type background recreates a brx phenotype. Paradoxically, however, while cvp2 or cvl1 single mutants display no apparent root defects, the root phenotype of cvp2 cvl1 double mutants is similar to brx or ops, although, as expected, cvp2 cvl1 seedlings contain more phosphatidylinositol-4,5-biphosphate. Thus, tightly balanced phosphatidylinositol-4,5-biphosphate levels appear essential for proper protophloem differentiation. Genetically, OPS acts downstream of phosphatidylinositol-4,5-biphosphate levels, as cvp2 mutation cannot rescue ops defects, whereas increased OPS dose rescues cvp2 cvl1 defects. Finally, all three mutants display higher density and accelerated emergence of lateral roots, which correlates with increased auxin response in the root differentiation zone. This phenotype is also created by application of peptides that suppress protophloem differentiation, CLAVATA3/EMBRYO SURROUNDING REGION 26 (CLE26) and CLE45. Thus, local changes in the primary root protophloem systemically shape overall root system architecture. HIGHLIGHTED ARTICLE: Protophloem differentiation in the Arabidopsisroot meristem requires locally balanced phosphoinositide levels, the disturbance of which impairs protophloem development and re-shapes root architecture.

Positional Information by Differential Endocytosis Splits Auxin Response to Drive Arabidopsis Root Meristem Growth

In the Arabidopsis root meristem, polar auxin transport creates a transcriptional auxin response gradient that peaks at the stem cell niche and gradually decreases as stem cell daughters divide and differentiate [1-3]. The amplitude and extent of this gradient are essential for both stem cell maintenance and root meristem growth [4, 5]. To investigate why expression of some auxin-responsive genes, such as the essential root meristem growth regulator BREVIS RADIX (BRX) [6], deviates from this gradient, we combined experimental and computational approaches. We created cellular-level root meristem models that accurately reproduce distribution of nuclear auxin activity and allow dynamic modeling of regulatory processes to guide experimentation. Expression profiles deviating from the auxin gradient could only be modeled after intersection of auxin activity with the observed differential endocytosis pattern and positive autoregulatory feedback through plasma-membrane-to-nucleus transfer of BRX. Because BRX is required for expression of certain auxin response factor targets, our data suggest a cell-type-specific endocytosis-dependent input into transcriptional auxin perception. This input sustains expression of a subset of auxin-responsive genes across the root meristems division and transition zones and is essential for meristem growth. Thus, the endocytosis pattern provides specific positional information to modulate auxin response.

A root specific induction of carotenoid biosynthesis contributes to ABA production upon salt stress in arabidopsis.

Abscisic acid (ABA) is a hormone that plays a vital role in mediating abiotic stress responses in plants. Salt exposure induces the synthesis of ABA through the cleavage of carotenoid precursors (xanthophylls), which are found at very low levels in roots. Here we show that de novo ABA biosynthesis in salt-treated Arabidopsis thaliana roots involves an organ-specific induction of the carotenoid biosynthetic pathway. Upregulation of the genes encoding phytoene synthase (PSY) and other enzymes of the pathway producing ABA precursors was observed in roots but not in shoots after salt exposure. A pharmacological block of the carotenoid pathway substantially reduced ABA levels in stressed roots, confirming that an increase in carotenoid accumulation contributes to fuel hormone production after salt exposure. Treatment with exogenous ABA was also found to upregulate PSY expression only in roots, suggesting an organ-specific feedback regulation of the carotenoid pathway by ABA. Taken together, our results show that the presence of high concentrations of salt in the growth medium rapidly triggers a root-specific activation of the carotenoid pathway, probably to ensure a proper supply of ABA precursors required for a sustained production of the hormone.

Colors in the dark: a model for the regulation of carotenoid biosynthesis in etioplasts.

Carotenoids are plastidial isoprenoid pigments essential for plant life. High carotenoid levels are found in chloroplasts and chromoplasts, but they are also produced in the etioplasts of seedlings that germinate in the dark. Our recent work has shown that an enhanced production of carotenoids in plastids of dark-grown Arabidopsis thaliana seedlings results in an improved transition to photosynthetic development (greening) upon illumination, illustrating the relevance of regulating etioplast carotenoid biosynthesis for plant fitness. We showed that the biosynthesis of etioplast carotenoids is controlled at the level of phytoene synthase (PSY), the enzyme catalyzing the first committed step of the pathway. Upregulation of PSY is necessary and sufficient to increase the production of carotenoids in dark-grown seedlings, in part because it triggers a feedback mechanism leading to the post-transcriptional accumulation of flux-controlling enzymes of the methylerythritol 4-phosphate (MEP) pathway, which synthesizes the substrates for PSY activity. Based on these and other recent data on the molecular mechanisms controlling deetiolation, we propose a model for the regulation of carotenoid biosynthesis in etioplasts.

Context-Dependent Dual Role of SKI8 Homologs in mRNA Synthesis and Turnover

Eukaryotic mRNA transcription and turnover is controlled by an enzymatic machinery that includes RNA polymerase II and the 3′ to 5′ exosome. The activity of these protein complexes is modulated by additional factors, such as the nuclear RNA polymerase II-associated factor 1 (Paf1c) and the cytoplasmic Superkiller (SKI) complex, respectively. Their components are conserved across uni- as well as multi-cellular organisms, including yeast, Arabidopsis, and humans. Among them, SKI8 displays multiple facets on top of its cytoplasmic role in the SKI complex. For instance, nuclear yeast ScSKI8 has an additional function in meiotic recombination, whereas nuclear human hSKI8 (unlike ScSKI8) associates with Paf1c. The Arabidopsis SKI8 homolog VERNALIZATION INDEPENDENT 3 (VIP3) has been found in Paf1c as well; however, whether it also has a role in the SKI complex remains obscure so far. We found that transgenic VIP3-GFP, which complements a novel vip3 mutant allele, localizes to both nucleus and cytoplasm. Consistently, biochemical analyses suggest that VIP3–GFP associates with the SKI complex. A role of VIP3 in the turnover of nuclear encoded mRNAs is supported by random-primed RNA sequencing of wild-type and vip3 seedlings, which indicates mRNA stabilization in vip3. Another SKI subunit homolog mutant, ski2, displays a dwarf phenotype similar to vip3. However, unlike vip3, it displays neither early flowering nor flower development phenotypes, suggesting that the latter reflect VIP3s role in Paf1c. Surprisingly then, transgenic ScSKI8 rescued all aspects of the vip3 phenotype, suggesting that the dual role of SKI8 depends on species-specific cellular context.

Wiring a plant: genetic networks for phloem formation in Arabidopsis thaliana roots

In plants, phloem conduits form a specialized vascular network mediating the exchange of nutrients and signaling molecules between distantly separated organs. To become effective transport elements, protophloem cells undergo a rather unique, differentiation program that involves nucleus degradation, organelle rearrangement and cell wall thickening. Yet, protophloem sieve elements remain alive because their essential metabolic functions are supported by their neighboring companion cells. In spite of the importance of the phloem, the molecular mechanisms orchestrating protophloem specification and differentiation remain still poorly understood. In this review, I provide a summary of recent discoveries regarding morphogenetic events that determine phloem formation, and also a discussion of the systemic effects on root architecture derived from impaired protophloem differentiation programs.

Perception of root-active CLE peptides requires CORYNE function in the phloem vasculature

Arabidopsis root development is orchestrated by signaling pathways that consist of different CLAVATA3/EMBRYO SURROUNDING REGION (CLE) peptide ligands and their cognate CLAVATA (CLV) and BARELY ANY MERISTEM (BAM) receptors. How and where different CLE peptides trigger specific morphological or physiological changes in the root is poorly understood. Here, we report that the receptor‐like protein CLAVATA 2 (CLV2) and the pseudokinase CORYNE (CRN) are necessary to fully sense root‐active CLE peptides. We uncover BAM3 as the CLE45 receptor in the root and biochemically map its peptide binding surface. In contrast to other plant peptide receptors, we found no evidence that SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) proteins act as co‐receptor kinases in CLE45 perception. CRN stabilizes BAM3 expression and thus is required for BAM3‐mediated CLE45 signaling. Moreover, protophloem‐specific CRN expression complements resistance of the crn mutant to root‐active CLE peptides, suggesting that protophloem is their principal site of action. Our work defines a genetic framework for dissecting CLE peptide signaling and CLV/BAM receptor activation in the root.