MFP1 defines the subchloroplast location of starch granule initiation.

Starch is one of the major carbohydrate storage compounds in plants. The biogenesis of starch granules starts with the formation of initials, which subsequently expand into granules. Several coiled-coil domain-containing proteins have been previously implicated with the initiation process, but the mechanisms by which they act remain largely elusive. Here, we demonstrate that one of these proteins, the thylakoid-associated MAR-BINDING FILAMENT-LIKE PROTEIN 1 (MFP1), specifically determines the subchloroplast location of initial formation. The expression of MFP1 variants "mis"-targeted to specific locations within chloroplasts in Arabidopsis results in distinctive shifts in not only how many but also where starch granules are formed. Importantly, "re" localizing MFP1 to the stromal face of the chloroplast's inner envelope is sufficient to generate starch granules in this aberrant position. These findings provide compelling evidence that a single protein MFP1 possesses the capacity to direct the initiation and biosynthesis machinery of starch granules.

Plasmodesmal connectivity in C4 Gynandropsis gynandra is induced by light and dependent on photosynthesis.

In leaves of C4 plants, the reactions of photosynthesis become restricted between two compartments. Typically, this allows accumulation of C4 acids in mesophyll (M) cells and subsequent decarboxylation in the bundle sheath (BS). In C4 grasses, proliferation of plasmodesmata between these cell types is thought to increase cell-to-cell connectivity to allow efficient metabolite movement. However, it is not known whether C4 dicotyledons also show this enhanced plasmodesmal connectivity and so whether this is a general requirement for C4 photosynthesis is not clear. How M and BS cells in C4 leaves become highly connected is also not known. We investigated these questions using 3D- and 2D-electron microscopy on the C4 dicotyledon Gynandropsis gynandra as well as phylogenetically close C3 relatives. The M-BS interface of C4 G. gynandra showed higher plasmodesmal frequency compared with closely related C3 species. Formation of these plasmodesmata was induced by light. Pharmacological agents that perturbed photosynthesis reduced the number of plasmodesmata, but this inhibitory effect could be reversed by the provision of exogenous sucrose. We conclude that enhanced formation of plasmodesmata between M and BS cells is wired to the induction of photosynthesis in C4 G. gynandra.

STARCH SYNTHASE5, a Noncanonical Starch Synthase-Like Protein, Promotes Starch Granule Initiation in Arabidopsis.

What determines the number of starch granules in plastids is an enigmatic aspect of starch metabolism. Several structurally and functionally diverse proteins have been implicated in the granule initiation process in Arabidopsis (Arabidopsis thaliana), with each protein exerting a varying degree of influence. Here, we show that a conserved starch synthase-like protein, STARCH SYNTHASE5 (SS5), regulates the number of starch granules that form in Arabidopsis chloroplasts. Among the starch synthases, SS5 is most closely related to SS4, a major determinant of granule initiation and morphology. However, unlike SS4 and the other starch synthases, SS5 is a noncanonical isoform that lacks catalytic glycosyltransferase activity. Nevertheless, loss of SS5 reduces starch granule numbers that form per chloroplast in Arabidopsis, and ss5 mutant starch granules are larger than wild-type granules. Like SS4, SS5 has a conserved putative surface binding site for glucans and also interacts with MYOSIN-RESEMBLING CHLOROPLAST PROTEIN, a proposed structural protein influential in starch granule initiation. Phenotypic analysis of a suite of double mutants lacking both SS5 and other proteins implicated in starch granule initiation allows us to propose how SS5 may act in this process.

Ectopic maltase alleviates dwarf phenotype and improves plant frost tolerance of maltose transporter mutants.

Maltose, the major product of starch breakdown in Arabidopsis (Arabidopsis thaliana) leaves, exits the chloroplast via the maltose exporter1 MEX1. Consequently, mex1 loss-of-function plants exhibit substantial maltose accumulation, a starch-excess phenotype and a specific chlorotic phenotype during leaf development. Here, we investigated whether the introduction of an alternative metabolic route could suppress the marked developmental defects typical for mex1 loss-of-function mutants. To this end, we ectopically expressed in mex1  chloroplasts a functional maltase (MAL) from baker's yeast (Saccharomyces cerevisiae, chloroplastidial MAL [cpMAL] mutants). Remarkably, the stromal MAL activity substantially alleviates most phenotypic peculiarities typical for mex1 plants. However, the cpMAL lines contained only slightly less maltose than parental mex1 plants and their starch levels were, surprisingly, even higher. These findings point to a threshold level of maltose responsible for the marked developmental defects in mex1. While growth and flowering time were only slightly retarded, cpMAL lines exhibited a substantially improved frost tolerance, when compared to wild-types. In summary, these results demonstrate the possibility to bypass the MEX1 transporter, allow us to differentiate between possible starch-excess and maltose-excess responses, and demonstrate that stromal maltose accumulation prevents frost defects. The latter insight may be instrumental for the development of crop plants with improved frost tolerance.

LIKE SEX4 1 Acts as a ß-Amylase-Binding Scaffold on Starch Granules during Starch Degradation.

In Arabidopsis (Arabidopsis thaliana) leaves, starch is synthesized during the day and degraded at night to fuel growth and metabolism. Starch is degraded primarily by ß-amylases, liberating maltose, but this activity is preceded by glucan phosphorylation and is accompanied by dephosphorylation. A glucan phosphatase family member, LIKE SEX4 1 (LSF1), binds starch and is required for normal starch degradation, but its exact role is unclear. Here, we show that LSF1 does not dephosphorylate glucans. The recombinant dual specificity phosphatase (DSP) domain of LSF1 had no detectable phosphatase activity. Furthermore, a variant of LSF1 mutated in the catalytic cysteine of the DSP domain complemented the starch-excess phenotype of the lsf1 mutant. By contrast, a variant of LSF1 with mutations in the carbohydrate binding module did not complement lsf1 Thus, glucan binding, but not phosphatase activity, is required for the function of LSF1 in starch degradation. LSF1 interacts with the ß-amylases BAM1 and BAM3, and the BAM1-LSF1 complex shows amylolytic but not glucan phosphatase activity. Nighttime maltose levels are reduced in lsf1, and genetic analysis indicated that the starch-excess phenotype of lsf1 is dependent on bam1 and bam3 We propose that LSF1 binds ß-amylases at the starch granule surface, thereby promoting starch degradation.

Distinct plastid fructose bisphosphate aldolases function in photosynthetic and non-photosynthetic metabolism in Arabidopsis.

Plastid metabolism is critical in both photoautotrophic and heterotrophic plant cells. In chloroplasts, fructose-1,6-bisphosphate aldolase (FBA) catalyses the formation of both fructose 1,6-bisphosphate and sedoheptulose 1,7-bisphosphate within the Calvin-Benson cycle. Three Arabidopsis genes, AtFBA1-AtFBA3, encode plastidial isoforms of FBA, but the contribution of each isoform is unknown. Phylogenetic analysis indicates that FBA1 and FBA2 derive from a recently duplicated gene, while FBA3 is a more ancient paralog. fba1 mutants are phenotypically indistinguishable from the wild type, while both fba2 and fba3 have reduced growth. We show that FBA2 is the major isoform in leaves, contributing most of the measurable activity. Partial redundancy with FBA1 allows both single mutants to survive, but combining both mutations is lethal, indicating a block of photoautotrophy. In contrast, FBA3 is expressed predominantly in heterotrophic tissues, especially the leaf and root vasculature, but not in the leaf mesophyll. We show that the loss of FBA3 affects plastidial glycolytic metabolism of the root, potentially limiting the biosynthesis of essential compounds such as amino acids. However, grafting experiments suggest that fba3 is dysfunctional in leaf phloem transport, and we suggest that a block in photoassimilate export from leaves causes the buildup of high carbohydrate concentrations and retarded growth.

Two Plastidial Coiled-Coil Proteins Are Essential for Normal Starch Granule Initiation in Arabidopsis.

The mechanism of starch granule initiation in chloroplasts is not fully understood. Here, we aimed to build on our recent discovery that PROTEIN TARGETING TO STARCH (PTST) family members, PTST2 and PTST3, are key players in starch granule initiation, by identifying and characterizing additional proteins involved in the process in Arabidopsis thaliana chloroplasts. Using immunoprecipitation and mass spectrometry, we demonstrate that PTST2 interacts with two plastidial coiled-coil proteins. Surprisingly, one of the proteins is the thylakoid-associated MAR BINDING FILAMENT-LIKE PROTEIN1 (MFP1), which was proposed to bind plastid nucleoids. The other protein, MYOSIN-RESEMBLING CHLOROPLAST PROTEIN (MRC), contains long coiled coils and no known domains. Whereas wild-type chloroplasts contained multiple starch granules, only one large granule was observed in most chloroplasts of the mfp1 and mrc mutants. The mfp1 mrc double mutant had a higher proportion of chloroplasts containing no visible granule than either single mutant and accumulated ADP-glucose, the substrate for starch synthesis. PTST2 was partially associated with the thylakoid membranes in wild-type plants, and fluorescently tagged PTST2 was located in numerous discrete patches within the chloroplast in which MFP1 was also located. In the mfp1 mutant, PTST2 was not associated with the thylakoids and formed discrete puncta, suggesting that MFP1 is necessary for normal PTST2 localization. Overall, we reveal that proper granule initiation requires the presence of MFP1 and MRC, and the correct location of PTST2.

Distinct Functions of STARCH SYNTHASE 4 Domains in Starch Granule Formation.

The formation of normal starch granules in Arabidopsis (Arabidopsis thaliana) leaf chloroplasts requires STARCH SYNTHASE 4 (SS4). In plants lacking SS4, chloroplasts typically produce only one round granule rather than multiple lenticular granules. The mechanisms by which SS4 determines granule number and morphology are not understood. The N-terminal region of SS4 is unique among SS isoforms and contains several long coiled-coil motifs, typically implicated in protein-protein interactions. The C-terminal region contains the catalytic glucosyltransferase domains, which are widely conserved in plant SS and bacterial glycogen synthase (GS) isoforms. We investigated the specific roles of the N- and C-terminal regions of SS4 by expressing truncated versions of SS4 and a fusion between the N-terminal region of SS4 and GS in the Arabidopsis ss4 mutant. Expression of the N-terminal region of SS4 alone did not alter the ss4 mutant phenotype. Expression of the C-terminal region of SS4 alone increased granule initiation but did not rescue their aberrant round morphology. Expression of a self-priming GS from Agrobacterium tumefaciens also increased the number of round granules. Remarkably, fusion of the N-terminal region of SS4 to A. tumefaciens GS restored the development of wild-type-like lenticular starch granules. Interestingly, the N-terminal region of SS4 alone or when fused to GS conferred a patchy subchloroplastic localization similar to that of the full-length SS4 protein. Considered together, these data suggest that, while the glucosyltransferase activity of SS4 is important for granule initiation, the N-terminal part of SS4 serves to establish the correct granule morphology by properly localizing this activity.

The Starch Granule-Associated Protein EARLY STARVATION1 Is Required for the Control of Starch Degradation in Arabidopsis thaliana Leaves.

To uncover components of the mechanism that adjusts the rate of leaf starch degradation to the length of the night, we devised a screen for mutant Arabidopsis thaliana plants in which starch reserves are prematurely exhausted. The mutation in one such mutant, named early starvation1 (esv1), eliminates a previously uncharacterized protein. Starch in mutant leaves is degraded rapidly and in a nonlinear fashion, so that reserves are exhausted 2 h prior to dawn. The ESV1 protein and a similar uncharacterized Arabidopsis protein (named Like ESV1 [LESV]) are located in the chloroplast stroma and are also bound into starch granules. The region of highest similarity between the two proteins contains a series of near-repeated motifs rich in tryptophan. Both proteins are conserved throughout starch-synthesizing organisms, from angiosperms and monocots to green algae. Analysis of transgenic plants lacking or overexpressing ESV1 or LESV, and of double mutants lacking ESV1 and another protein necessary for starch degradation, leads us to propose that these proteins function in the organization of the starch granule matrix. We argue that their misexpression affects starch degradation indirectly, by altering matrix organization and, thus, accessibility of starch polymers to starch-degrading enzymes.

PROTEIN TARGETING TO STARCH is required for localising GRANULE-BOUND STARCH SYNTHASE to starch granules and for normal amylose synthesis in Arabidopsis.

The domestication of starch crops underpinned the development of human civilisation, yet we still do not fully understand how plants make starch. Starch is composed of glucose polymers that are branched (amylopectin) or linear (amylose). The amount of amylose strongly influences the physico-chemical behaviour of starchy foods during cooking and of starch mixtures in non-food manufacturing processes. The GRANULE-BOUND STARCH SYNTHASE (GBSS) is the glucosyltransferase specifically responsible for elongating amylose polymers and was the only protein known to be required for its biosynthesis. Here, we demonstrate that PROTEIN TARGETING TO STARCH (PTST) is also specifically required for amylose synthesis in Arabidopsis. PTST is a plastidial protein possessing an N-terminal coiled coil domain and a C-terminal carbohydrate binding module (CBM). We discovered that Arabidopsis ptst mutants synthesise amylose-free starch and are phenotypically similar to mutants lacking GBSS. Analysis of granule-bound proteins showed a dramatic reduction of GBSS protein in ptst mutant starch granules. Pull-down assays with recombinant proteins in vitro, as well as immunoprecipitation assays in planta, revealed that GBSS physically interacts with PTST via a coiled coil. Furthermore, we show that the CBM domain of PTST, which mediates its interaction with starch granules, is also required for correct GBSS localisation. Fluorescently tagged Arabidopsis GBSS, expressed either in tobacco or Arabidopsis leaves, required the presence of Arabidopsis PTST to localise to starch granules. Mutation of the CBM of PTST caused GBSS to remain in the plastid stroma. PTST fulfils a previously unknown function in targeting GBSS to starch. This sheds new light on the importance of targeting biosynthetic enzymes to sub-cellular sites where their action is required. Importantly, PTST represents a promising new gene target for the biotechnological modification of starch composition, as it is exclusively involved in amylose synthesis.

Increasing the carbohydrate storage capacity of plants by engineering a glycogen-like polymer pool in the cytosol.

Global demand for higher crop yields and for more efficient utilization of agricultural products will grow over the next decades. Here, we present a new concept for boosting the carbohydrate content of plants, by channeling photosynthetically fixed carbon into a newly engineered glucose polymer pool. We transiently expressed the starch/glycogen synthases from either Saccharomyces cerevisiae or Cyanidioschyzon merolae, together with the starch branching enzyme from C. merolae, in the cytosol of Nicotiana benthamiana leaves. This effectively built a UDP-glucose-dependent glycogen biosynthesis pathway. Glycogen synthesis was observed with Transmission Electron Microscopy, and the polymer structure was further analyzed. Within three days of enzyme expression, glycogen content of the leaf was 5-10 times higher than the starch levels of the control. Further, the leaves produced less starch and sucrose, which are normally the carbohydrate end-products of photosynthesis. We conclude that after enzyme expression, the newly fixed carbohydrates were routed into the new glycogen sink and trapped. Our approach allows carbohydrates to be efficiently stored in a new subcellular compartment, thus increasing the value of vegetative crop tissues for biofuel production or animal feed. The method also opens new potential for increasing the sink strength of heterotrophic tissues.

Cecropia peltata accumulates starch or soluble glycogen by differentially regulating starch biosynthetic genes.

The branched glucans glycogen and starch are the most widespread storage carbohydrates in living organisms. The production of semicrystalline starch granules in plants is more complex than that of small, soluble glycogen particles in microbes and animals. However, the factors determining whether glycogen or starch is formed are not fully understood. The tropical tree Cecropia peltata is a rare example of an organism able to make either polymer type. Electron micrographs and quantitative measurements show that glycogen accumulates to very high levels in specialized myrmecophytic structures (Müllerian bodies), whereas starch accumulates in leaves. Compared with polymers comprising leaf starch, glycogen is more highly branched and has shorter branches--factors that prevent crystallization and explain its solubility. RNA sequencing and quantitative shotgun proteomics reveal that isoforms of all three classes of glucan biosynthetic enzyme (starch/glycogen synthases, branching enzymes, and debranching enzymes) are differentially expressed in Müllerian bodies and leaves, providing a system-wide view of the quantitative programming of storage carbohydrate metabolism. This work will prompt targeted analysis in model organisms and cross-species comparisons. Finally, as starch is the major carbohydrate used for food and industrial applications worldwide, these data provide a basis for manipulating starch biosynthesis in crops to synthesize tailor-made polyglucans.

Genetic Evidence That Chain Length and Branch Point Distributions Are Linked Determinants of Starch Granule Formation in Arabidopsis.

The major component of starch is the branched glucan amylopectin. Structural features of amylopectin, such as the branching pattern and the chain length distribution, are thought to be key factors that enable it to form semicrystalline starch granules. We varied both structural parameters by creating Arabidopsis (Arabidopsis thaliana) mutants lacking combinations of starch synthases (SSs) SS1, SS2, and SS3 (to vary chain lengths) and the debranching enzyme ISOAMYLASE1-ISOAMYLASE2 (ISA; to alter branching pattern). The isa mutant accumulates primarily phytoglycogen in leaf mesophyll cells, with only small amounts of starch in other cell types (epidermis and bundle sheath cells). This balance can be significantly shifted by mutating different SSs. Mutation of SS1 promoted starch synthesis, restoring granules in mesophyll cell plastids. Mutation of SS2 decreased starch synthesis, abolishing granules in epidermal and bundle sheath cells. Thus, the types of SSs present affect the crystallinity and thus the solubility of the glucans made, compensating for or compounding the effects of an aberrant branching pattern. Interestingly, ss2 mutant plants contained small amounts of phytoglycogen in addition to aberrant starch. Likewise, ss2ss3 plants contained phytoglycogen, but were almost devoid of glucan despite retaining other SS isoforms. Surprisingly, glucan production was restored in the ss2ss3isa triple mutants, indicating that SS activity in ss2ss3 per se is not limiting but that the isoamylase suppresses glucan accumulation. We conclude that loss of only SSs can cause phytoglycogen production. This is readily degraded by isoamylase and other enzymes so it does not accumulate and was previously unnoticed.

Plastidial NAD-dependent malate dehydrogenase is critical for embryo development and heterotrophic metabolism in Arabidopsis.

In illuminated chloroplasts, one mechanism involved in reduction-oxidation (redox) homeostasis is the malate-oxaloacetate (OAA) shuttle. Excess electrons from photosynthetic electron transport in the form of nicotinamide adenine dinucleotide phosphate, reduced are used by NADP-dependent malate dehydrogenase (MDH) to reduce OAA to malate, thus regenerating the electron acceptor NADP. NADP-MDH is a strictly redox-regulated, light-activated enzyme that is inactive in the dark. In the dark or in nonphotosynthetic tissues, the malate-OAA shuttle was proposed to be mediated by the constitutively active plastidial NAD-specific MDH isoform (pdNAD-MDH), but evidence is scarce. Here, we reveal the critical role of pdNAD-MDH in Arabidopsis (Arabidopsis thaliana) plants. A pdnad-mdh null mutation is embryo lethal. Plants with reduced pdNAD-MDH levels by means of artificial microRNA (miR-mdh-1) are viable, but dark metabolism is altered as reflected by increased nighttime malate, starch, and glutathione levels and a reduced respiration rate. In addition, miR-mdh-1 plants exhibit strong pleiotropic effects, including dwarfism, reductions in chlorophyll levels, photosynthetic rate, and daytime carbohydrate levels, and disordered chloroplast ultrastructure, particularly in developing leaves, compared with the wild type. pdNAD-MDH deficiency in miR-mdh-1 can be functionally complemented by expression of a microRNA-insensitive pdNAD-MDH but not NADP-MDH, confirming distinct roles for NAD- and NADP-linked redox homeostasis.

The simultaneous abolition of three starch hydrolases blocks transient starch breakdown in Arabidopsis.

In this study, we investigated which enzymes are involved in debranching amylopectin during transient starch degradation. Previous studies identified two debranching enzymes, isoamylase 3 (ISA3) and limit dextrinase (LDA), involved in this process. However, plants lacking both enzymes still degrade substantial amounts of starch. Thus, other enzymes/mechanisms must contribute to starch breakdown. We show that the chloroplastic α-amylase 3 (AMY3) also participates in starch degradation and provide evidence that all three enzymes can act directly at the starch granule surface. The isa3 mutant has a starch excess phenotype, reflecting impaired starch breakdown. In contrast, removal of AMY3, LDA, or both enzymes together has no impact on starch degradation. However, removal of AMY3 or LDA in addition to ISA3 enhances the starch excess phenotype. In plants lacking all three enzymes, starch breakdown is effectively blocked, and starch accumulates to the highest levels observed so far. This provides indirect evidence that the heteromultimeric debranching enzyme ISA1-ISA2 is not involved in starch breakdown. However, we illustrate that ISA1-ISA2 can hydrolyze small soluble branched glucans that accumulate when ISA3 and LDA are missing, albeit at a slow rate. Starch accumulation in the mutants correlates inversely with plant growth.

Starch synthase 4 is essential for coordination of starch granule formation with chloroplast division during Arabidopsis leaf expansion.

Arabidopsis thaliana mutants lacking the SS4 isoform of starch synthase have strongly reduced numbers of starch granules per chloroplast, suggesting that SS4 is necessary for the normal generation of starch granules. To establish whether it plays a direct role in this process, we investigated the circumstances in which granules are formed in ss4 mutants. Starch granule numbers and distribution and the accumulation of starch synthase substrates and products were investigated during ss4 leaf development, and in ss4 mutants carrying mutations or transgenes that affect starch turnover or chloroplast volume. We found that immature ss4 leaves have no starch granules, but accumulate high concentrations of the starch synthase substrate ADPglucose. Granule numbers are partially restored by elevating the capacity for glucan synthesis (via expression of bacterial glycogen synthase) or by increasing the volumes of individual chloroplasts (via introduction of arc mutations). However, these granules are abnormal in distribution, size and shape. SS4 is an essential component of a mechanism that coordinates granule formation with chloroplast division during leaf expansion and determines the abundance and the flattened, discoid shape of leaf starch granules.

LIKE EARLY STARVATION 1 and EARLY STARVATION 1 promote and stabilize amylopectin phase transition in starch biosynthesis.

Starch, the most abundant carbohydrate reserve in plants, primarily consists of the branched glucan amylopectin, which forms semi-crystalline granules. Phase transition from a soluble to an insoluble form depends on amylopectin architecture, requiring a compatible distribution of glucan chain lengths and a branch-point distribution. Here, we show that two starch-bound proteins, LIKE EARLY STARVATION 1 (LESV) and EARLY STARVATION 1 (ESV1), which have unusual carbohydrate-binding surfaces, promote the phase transition of amylopectin-like glucans, both in a heterologous yeast system expressing the starch biosynthetic machinery and in Arabidopsis plants. We propose a model wherein LESV serves as a nucleating role, with its carbohydrate-binding surfaces helping align glucan double helices to promote their phase transition into semi-crystalline lamellae, which are then stabilized by ESV1. Because both proteins are widely conserved, we suggest that protein-facilitated glucan crystallization may be a general and previously unrecognized feature of starch biosynthesis.

Coalescence and directed anisotropic growth of starch granule initials in subdomains of Arabidopsis thaliana chloroplasts.

Living cells orchestrate enzyme activities to produce myriads of biopolymers but cell-biological understanding of such processes is scarce. Starch, a plant biopolymer forming discrete, semi-crystalline granules within plastids, plays a central role in glucose storage, which is fundamental to life. Combining complementary imaging techniques and Arabidopsis genetics we reveal that, in chloroplasts, multiple starch granules initiate in stromal pockets between thylakoid membranes. These initials coalesce, then grow anisotropically to form lenticular granules. The major starch polymer, amylopectin, is synthesized at the granule surface, while the minor amylose component is deposited internally. The non-enzymatic domain of STARCH SYNTHASE 4, which controls the protein's localization, is required for anisotropic growth. These results present us with a conceptual framework for understanding the biosynthesis of this key nutrient.

A multifaceted analysis reveals two distinct phases of chloroplast biogenesis during de-etiolation in Arabidopsis.

Light triggers chloroplast differentiation whereby the etioplast transforms into a photosynthesizing chloroplast and the thylakoid rapidly emerges. However, the sequence of events during chloroplast differentiation remains poorly understood. Using Serial Block Face Scanning Electron Microscopy (SBF-SEM), we generated a series of chloroplast 3D reconstructions during differentiation, revealing chloroplast number and volume and the extent of envelope and thylakoid membrane surfaces. Furthermore, we used quantitative lipid and whole proteome data to complement the (ultra)structural data, providing a time-resolved, multi-dimensional description of chloroplast differentiation. This showed two distinct phases of chloroplast biogenesis: an initial photosynthesis-enabling 'Structure Establishment Phase' followed by a 'Chloroplast Proliferation Phase' during cell expansion. Moreover, these data detail thylakoid membrane expansion during de-etiolation at the seedling level and the relative contribution and differential regulation of proteins and lipids at each developmental stage. Altogether, we establish a roadmap for chloroplast differentiation, a critical process for plant photoautotrophic growth and survival.

A Reservoir of Pluripotent Phloem Cells Safeguards the Linear Developmental Trajectory of Protophloem Sieve Elements.

Plant cells can change their identity based on positional information, a mechanism that confers developmental plasticity to plants. This ability, common to distinct multicellular organisms, is particularly relevant for plant phloem cells. Protophloem sieve elements (PSEs), one type of phloem conductive cells, act as the main organizers of the phloem pole, which comprises four distinct cell files organized in a conserved pattern. Here, we report how Arabidopsis roots generate a reservoir of meristematic phloem cells competent to swap their cell identities. Although PSE misspecification induces cell identity hybridism, the activity of RECEPTOR LIKE PROTEIN KINASE 2 (RPK2) by perceiving CLE45 peptide contributes to restrict PSE identity to the PSE position. By maintaining a spatiotemporal window when PSE and PSE-adjacent cells' identities are interchangeable, CLE45 signaling endows phloem cells with the competence to re-pattern a functional phloem pole when protophloem fails to form.