SUMMARYPlant growth is strongly influenced by the presence of neighbors that compete for light resources. In response to vegetational shading shade-intolerant plants such as Arabidopsis display a suite of developmental responses known as the shade-avoidance syndrome (SAS). The phytochrome B (phyB) photoreceptor is the major light sensor to mediate this adaptive response. Control of the SAS occurs in part with phyB, which controls protein abundance of phytochrome-interacting factors 4 and 5 (PIF4 and PIF5) directly. The shadeavoidance response also requires rapid biosynthesis of auxin and its transport to promote elongation growth. The identification of genome-wide PIF5-binding sites during shade avoidance revealed that this bHLH transcription factor regulates the expression of a subset of previously identified SAS genes. Moreover our study suggests that PIF4 and PIF5 regulate elongation growth by controlling directly the expression of genes that code for auxin biosynthesis and auxin signaling components.

A previously unknown maltose transporter is essential for the conversion of starch to sucrose in Arabidopsis leaves at night. The transporter was identified by isolating two allelic mutants with high starch levels and very high maltose, an intermediate of starch breakdown. The mutations affect a gene of previously unknown function, MEX1. We show that MEX1is a maltose transporter that is unrelated to other sugar transporters. The severe mex1 phenotype demonstrates that MEX1is the predominant route of carbohydrate export from chloroplasts at night. Homologous genes in plants including rice and potato indicate that maltose export is of widespread significance.

ORCID IDs: 0000-0001-5075-575X (M.V.K.); 0000-0003-1339-5120 (E.S.-S.); 0000-0002-4145-7205 (F.S.); 0000-0001-9237-1797 (P.M.-M.); 0000-0002-9623-2599 (J.M.); 0000-0002-3413-6841 (I.X.); 0000-0003-4719-5901 (C.F.)In response to neighbor proximity, plants increase the growth of specific organs (e.g., hypocotyls) to enhance access to sunlight. Shade enhances the activity of Phytochrome Interacting Factors (PIFs) by releasing these bHLH transcription factors from phytochrome B-mediated inhibition. PIFs promote elongation by inducing auxin production in cotyledons. In order to elucidate spatiotemporal aspects of the neighbor proximity response, we separately analyzed gene expression patterns in the major light-sensing organ (cotyledons) and in rapidly elongating hypocotyls of Arabidopsis thaliana. PIFs initiate transcriptional reprogramming in both organs within 15 min, comprising regulated expression of several early auxin response genes. This suggests that hypocotyl growth is elicited by both local and distal auxin signals. We show that cotyledon-derived auxin is both necessary and sufficient to initiate hypocotyl growth, but we also provide evidence for the functional importance of the local PIFinduced response. With time, the transcriptional response diverges increasingly between organs. We identify genes whose differential expression may underlie organ-specific elongation. Finally, we uncover a growth promotion gene expression signature shared between different developmentally regulated growth processes and responses to the environment in different organs.

SummaryThe aim of this work was to evaluate the function of isoamylase in starch granule biosynthesis in Arabidopsis leaves. A reverse-genetic approach was used to knockout AtISA1, one of three genes in Arabidopsis encoding isoamylase-type debranching enzymes. The mutant (Atisa1-1) lacks functional AtISA1 transcript and the major isoamylase activity (detected by native gels) in crude extracts of leaves. The same activity is abolished by mutation at the DBE1 locus, which encodes a second isoamylase-type protein, AtISA2. This is consistent with the idea that ISA1 and ISA2 proteins are subunits of the same enzyme in vivo. Atisa1-1, Atisa2-1 (dbe1), and the Atisa1-1/Atisa2-1 double mutant all have identical phenotypes. Starch content is reduced compared with the wild type but substantial quantities of the soluble glucan phytoglycogen are produced. The amylopectin of the remaining starch and the phytoglycogen in the mutants are structurally related to each other and differ from wild-type amylopectin. Electron micrographs reveal that the phytoglycogen-accumulating phenotype is highly tissue-specific. Phytoglycogen accumulates primarily in the plastids of the palisade and spongy mesophyll cells. Remarkably, other cell types appear to accumulate only starch, which is normal in appearance but is altered in structure. As phytoglycogen accumulates during the day, its rate of accumulation decreases, its structure changes and intermediates of glucan breakdown accumulate, suggesting that degradation occurs simultaneously with synthesis. We conclude that the AtISA1/AtISA2 isoamylase influences glucan branching pattern, but that this may not be the primary determinant of partitioning between crystalline starch and soluble phytoglycogen.

Phototropism, or plant growth in response to unidirectional light, is an adaptive response of crucial importance. Lateral differences in low fluence rates of blue light are detected by phototropin 1 (phot1) in Arabidopsis. Only NONPHOTOTROPIC HYPOCOTYL 3 (NPH3) and root phototropism 2, both belonging to the same family of proteins, have been previously identified as phototropininteracting signal transducers involved in phototropism. PHYTO-CHROME KINASE SUBSTRATE (PKS) 1 and PKS2 are two phytochrome signaling components belonging to a small gene family in Arabidopsis (PKS1-PKS4). The strong enhancement of PKS1 expression by blue light and its light induction in the elongation zone of the hypocotyl prompted us to study the function of this gene family during phototropism. Photobiological experiments show that the PKS proteins are critical for hypocotyl phototropism. Furthermore, PKS1 interacts with phot1 and NPH3 in vivo at the plasma membrane and in vitro, indicating that the PKS proteins may function directly with phot1 and NPH3 to mediate phototropism. The phytochromes are known to influence phototropism but the mechanism involved is still unclear. We show that PKS1 induction by a pulse of blue light is phytochrome A-dependent, suggesting that the PKS proteins may provide a molecular link between these two photoreceptor families.Arabidopsis thaliana ͉ NONPHOTOTROPIC HYPOCOTYL 3 ͉ photomorphogenesis photoreceptors

Phototropic hypocotyl bending in response to blue light excitation is an important adaptive process that helps plants to optimize their exposure to light. In Arabidopsis thaliana, phototropic hypocotyl bending is initiated by the blue light receptors and protein kinases phototropin1 (phot1) and phot2. Phototropic responses also require auxin transport and were shown to be partially compromised in mutants of the PIN-FORMED (PIN) auxin efflux facilitators. We previously described the D6 PROTEIN KINASE (D6PK) subfamily of AGCVIII kinases, which we proposed to directly regulate PIN-mediated auxin transport. Here, we show that phototropic hypocotyl bending is strongly dependent on the activity of D6PKs and the PIN proteins PIN3, PIN4, and PIN7. While early blue light and phot-dependent signaling events are not affected by the loss of D6PKs, we detect a gradual loss of PIN3 phosphorylation in d6pk mutants of increasing complexity that is most severe in the d6pk d6pkl1 d6pkl2 d6pkl3 quadruple mutant. This is accompanied by a reduction of basipetal auxin transport in the hypocotyls of d6pk as well as in pin mutants. Based on our data, we propose that D6PK-dependent PIN regulation promotes auxin transport and that auxin transport in the hypocotyl is a prerequisite for phot1-dependent hypocotyl bending.

We report that protein phosphorylation is involved in the control of starch metabolism in Arabidopsis leaves at night. sex4 (starch excess 4) mutants, which have strongly reduced rates of starch metabolism, lack a protein predicted to be a dual specificity protein phosphatase. We have shown that this protein is chloroplastic and can bind to glucans and have presented evidence that it acts to regulate the initial steps of starch degradation at the granule surface. Remarkably, the most closely related protein to SEX4 outside the plant kingdom is laforin, a glucanbinding protein phosphatase required for the metabolism of the mammalian storage carbohydrate glycogen and implicated in a severe form of epilepsy (Lafora disease) in humans.Starch, the main storage carbohydrate of plants, accumulates as a product of photosynthesis in leaves during the day and is converted to sucrose for export from the leaves at night. This conversion of starch to sucrose is one of the largest daily carbon fluxes on the planet, but nothing is known about how the process is initiated and controlled. The amounts of enzymes on the pathway change very little through the diurnal cycle in leaves of the model plant Arabidopsis thaliana, hence flux must be controlled by modulation of their activities (1).Much progress in understanding the pathway has been made through the selection of Arabidopsis mutants impaired in starch degradation at night. All such mutations identified thus far are in genes encoding enzymes of the pathway, rather than proteins likely to be involved in modulation of the activities of these enzymes (2-11). However, a mutation at a locus not yet identified, the starch excess 4 (or SEX4) locus, gives rise to a phenotype indicative of a regulatory defect rather than a defect in a structural enzyme. Mature sex4 leaves contain three to four times more starch than those of wild-type plants, apparently because a reduced capacity for starch degradation at night leads to progressive accumulation of starch over the life of the leaf (12, 13). Starch granules in leaves of the sex4 mutant are much larger and more rounded than those of wild-type plants (14). Measurements of activity and protein of enzymes known to be involved in starch degradation revealed only one significant reduction in the sex4 mutant in the chloroplastic ␣-amylase AMY3 (12, 15). However, although both the activity and amount of protein of AMY3 are strongly reduced, this is not the cause of the deficiency in starch degradation in the sex4 mutant. T-DNA insertion lines lacking AMY3 protein have normal rates of starch degradation (15). The aim of the work described in this paper was to discover the nature of the gene at the SEX4 locus and thus shed light on the regulation of starch degradation. EXPERIMENTAL PROCEDURESPositional Identification of the SEX4 Locus-F2 plants from a cross between sex4-2 (Col-0 background) and Landsberg erecta showing the mutant phenotype were used for mapping. The mapping population (562 plants) was genotyped using SSLP and SNP markers availabl...

The aim of this work was to understand the initial steps of starch breakdown inside chloroplasts. In the non-living endosperm of germinating cereal grains, starch breakdown is initiated by ␣-amylase Here, we present evidence that the debranching enzyme isoamylase 3 (ISA3) acts at the surface of the starch granule. Atisa3 mutants have more leaf starch and a slower rate of starch breakdown than wild-type plants. The amylopectin of Atisa3 contains many very short branches and ISA3-GFP localizes to granule-like structures inside chloroplasts. We suggest that ISA3 removes short branches from the granule surface. To understand how some starch is still degraded in Atisa3 mutants we eliminated a second debranching enzyme, limit dextrinase (pullulanase-type). Atlda mutants are indistinguishable from the wild type. However, the Atisa3/Atlda double mutant has a more severe starch-excess phenotype and a slower rate of starch breakdown than Atisa3 single mutants. The double mutant accumulates soluble branched oligosaccharides (limit dextrins) that are undetectable in the wild-type and the single mutants. Together these results suggest that glucan debranching occurs primarily at the granule surface via ISA3, but in its absence soluble branched glucans are debranched in the stroma via limit dextrinase. Consistent with this model, chloroplastic ␣-amylase AtAMY3, which could release soluble branched glucans, is induced in Atisa3 and in the Atisa3/Atlda double mutant.Starch is the most abundant storage carbohydrate in plants. It is composed primarily of amylopectin, a branched polymer of glucose in which ␣-1,4-linked glucan chains are connected by ␣-1,6-bonds (branch points). In this way, amylopectin resembles glycogen, the soluble storage carbohydrate synthesized in prokaryotes, fungi, and animals. However, unlike glycogen, amylopectin molecules can form a semicrystalline granule. This capacity stems from the branching pattern of amylopectin, which produces clusters of linear chains that pack together in regular semicrystalline arrays. This gives rise to a macromolecular granule structure in which semicrystalline lamellae alternate with amorphous lamellae, where branch points are located (1, 2).The way in which the insoluble starch granule is degraded to release stored glucose is understood only in the endosperm of germinating cereals. In this tissue, the process of starch breakdown is initiated by the action of ␣-amylase, secreted into the non-living starchy endosperm (3). This enzyme has an endoamylolytic action and releases a mixture of branched and linear oligosaccharides that serve as substrates for other hydrolytic enzymes including debranching enzymes (which hydrolyze the ␣-1,6-bonds) and exoamylases (␤-amylases). There is mounting evidence that a different mechanism operates in living cells of other plant tissues, where the starch is degraded inside the plastid compartment. First, Arabidopsis mutants lacking all three ␣-amylases encoded in the genome have normal rates of starch breakdown under standard growthroom conditi...