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The organization of cortical microtubule arrays in higher plant cells is essential for organizing cell and tissue morphogenesis, but it is not clear how specific architectures are acquired and reconfigured in response to environmental cues. Lindeboom et al. ( 10.1126/science.1245533 , published online 7 November; see the Perspective by Roll-Mecak ) used live-cell imaging and genetic studies to show that the microtubule-severing protein, katanin, plays a crucial role in reorienting cortical arrays from transverse to longitudinal in Arabidopsis seedlings in response to blue light perception. Katanin localized to microtubule intersections where, stimulated by blue light receptors, it preferentially catalyzed the severing of the newer microtubule. The microtubule “plus” end created by severing were observed to grow preferentially, effectively building a new population of microtubules orthogonal to the initial array. The net effect of this process steers the growing seedling toward light. A self-organizing system makes the microtubule array in plants rearrange in order for the shoot to turn toward blue light. Environmental and hormonal signals cause reorganization of microtubule arrays in higher plants, but the mechanisms driving these transitions have remained elusive. The organization of these arrays is required to direct morphogenesis. We discovered that microtubule severing by the protein katanin plays a crucial and unexpected role in the reorientation of cortical arrays, as triggered by blue light. Imaging and genetic experiments revealed that phototropin photoreceptors stimulate katanin-mediated severing specifically at microtubule intersections, leading to the generation of new microtubules at these locations. We show how this activity serves as the basis for a mechanism that amplifies microtubules orthogonal to the initial array, thereby driving array reorientation. Our observations show how severing is used constructively to build a new microtubule array.

Lignin is an integral constituent of the primary cell walls of the dark-grown maize (Zea mays L.) coleoptile, a juvenile organ that is still in the developmental state of rapid cell extension. Coleoptile lignin was characterized by (i) conversion to lignothiolglycolate derivative, (ii) isolation of polymeric fragments after alkaline hydrolysis, (iii) reactivity to antibodies against dehydrogenative polymers prepared from monolignols, and (iv) identification of thioacidolysis products typical of lignins. Substantial amounts of lignin could be solubilized from the coleoptile cell walls by mild alkali treatments. Thioacidolysis analyses of cell walls from coleoptiles and various mesocotyl tissues demonstrated the presence of guaiacyl-, syringyl- and (traces of) p-hydroxyphenyl units besides p-coumaric and ferulic acids. There are tissue-specific differences in amount and composition of lignins from different parts of the maize seedling. Electron-microscopic immunogold labeling of epitopes recognized by a specific anti-guaiacyl/syringyl antibody demonstrated the presence of lignin in all cell walls of the 4-d-old coleoptile. The primary walls of parenchyma and epidermis were more weakly labeled than the secondary wall thickenings of tracheary elements. No label was found in middle lamellae and cell corners. Lignin epitopes appeared first in the tracheary elements on day 2 and in the parenchyma on day 3 after sowing. Incubation of coleoptile segments in H2O2 increased the amount of extractable lignin and the abundance of lignin epitopes in the parenchyma cell walls. Lignin deposition was temporally and spatially correlated with the appearance of epitopes for proline-rich proteins, but not for hydroxyproline-rich proteins, in the cell walls. The lignin content of coleoptiles was increased by irradiating the seedlings with white or farred light, correlated with the inhibition of elongation growth, while growth promotion by auxin had no effect. It is concluded that wall stiffness, and thus extension growth, of the coleoptile can be controlled by lignification of the primary cell walls. Primary-wall lignin may represent part of an extended polysaccharide-polyphenol network that limits the extensibility of the cell walls.

Plants are highly attuned to translating environmental changes to appropriate modifications in growth. Such phenotypic plasticity is observed in dense vegetations, where shading by neighboring plants, triggers rapid unidirectional shoot growth (shade avoidance), such as petiole elongation, which is partly under the control of auxin. This growth is fuelled by cellular expansion requiring cell-wall modification by proteins such as xyloglucan endotransglucosylase/hydrolases (XTHs). Cortical microtubules (cMTs) are highly dynamic cytoskeletal structures that are also implicated in growth regulation. The objective of this study was to investigate the tripartite interaction between auxin, cMTs and XTHs in shade avoidance. Our results indicate a role for cMTs to control rapid petiole elongation in Arabidopsis during shade avoidance. Genetic and pharmacological perturbation of cMTs obliterated shade-induced growth and led to a reduction in XTH activity as well. Furthermore, the cMT disruption repressed the shade-induced expression of a specific set of XTHs. These XTHs were also regulated by the hormone auxin, an important regulator of plant developmental plasticity and also of several shade avoidance responses. Accordingly, the effect of cMT disruption on the shade enhanced XTH expression could be rescued by auxin application. Based on the results we hypothesize that cMTs can mediate petiole elongation during shade avoidance by regulating the expression of cell wall modifying proteins via control of auxin distribution.

The plastic elongation of mesocotyl (MES) and coleoptile (COL), which can be repressed by light exposure, plays a vital role in maize seedling emergence and establishment under adverse environmental conditions. Understanding the molecular mechanisms of light-mediated repression of MES and COL elongation in maize will allow us to develop new strategies for genetic improvement of these two crucial traits in maize. A maize variety, Zheng58, was used to monitor the transcriptome and physiological changes in MES and COL in response to darkness, as well as red, blue, and white light. The elongation of MES and COL was significantly inhibited by light spectral quality in this order: blue light > red light > white light. Physiological analyses revealed that light-mediated inhibition of maize MES and COL elongation was closely related to the dynamics of phytohormones accumulation and lignin deposition in these tissues. In response to light exposure, the levels of indole-3-acetic acid, trans-zeatin, gibberellin 3, and abscisic acid levels significantly decreased in MES and COL; by contrast, the levels of jasmonic acid, salicylic acid, lignin, phenylalanine ammonia-lyase, and peroxidase enzyme activity significantly increased. Transcriptome analysis revealed multiple differentially expressed genes (DEGs) involved in circadian rhythm, phytohormone biosynthesis and signal transduction, cytoskeleton and cell wall organization, lignin biosynthesis, and starch and sucrose metabolism. These DEGs exhibited synergistic and antagonistic interactions, forming a complex network that regulated the light-mediated inhibition of MES and COL elongation. Additionally, gene co-expression network analysis revealed that 49 hub genes in one and 19 hub genes in two modules were significantly associated with the elongation plasticity of COL and MES, respectively. These findings enhance our knowledge of the light-regulated elongation mechanisms of MES and COL, and provide a theoretical foundation for developing elite maize varieties with improved abiotic stress resistance.

MAIN CONCLUSION: ZmPHOT1 and ZmPHOT2 are expressed differentially in maize coleoptiles and leaves, with Zmphot1 possibly involved in first-positive phototropic curvature of red-light-adapted maize coleoptiles exposed to pulsed low-fluence blue light. Unilateral blue-light perception by phototropin(s) is the first event of phototropism, with the subsequent signal causing lateral transport of auxin at the coleoptile tip region of monocots. In this study, we analyzed the behavior of two maize phototropin genes: ZmPHOT1 and ZmPHOT2, the latter identified from the maize genome database and newly characterized. Quantitative real-time PCR analysis demonstrated that ZmPHOT1 was abundantly expressed in etiolated coleoptiles, while lower expressions of both ZmPHOT1 and ZmPHOT2 were observed in young leaves. Interestingly, these genes were not specifically expressed in the coleoptile tip region, a key position for photoperception in phototropism. Exposure to pulsed low-fluence blue light (LBL) (0.33 µmol m⁻² s⁻¹ × 8 s) and continuous high-fluence blue light (HBL) (10 µmol m⁻² s⁻¹) rapidly decreased ZmPHOT1 gene expression in coleoptiles, with levels of ZmPHOT2 not significantly altered in that tissue. In young leaves, no drastic expression changes were induced in either ZmPHOT1 or ZmPHOT2 by LBL or HBL irradiation. The Zmphot1 protein was investigated by Western blot analysis with anti-Osphot1 antibodies. Zmphot1 was detected in microsomal fractions, with higher levels in coleoptiles than in leaves. HBL caused rapid phosphorylation of the protein, whereas no phot1 phosphorylation was induced by LBL. The involvement of Zmphot1 in LBL-induced phototropic curvature of maize coleoptiles is discussed.

Reactive oxygen intermediates, i.e. the superoxide radical (${\\mathrm{O}}_{2}^{\\cdot -}$), hydrogen peroxide (H2O2) and the hydroxyl radical (·OH), are generally regarded as harmful products of oxygenic metabolism causing cell damage in plants, animals and microorganisms. However, oxygen radical chemistry may also play a useful role in polymer breakdown leading to wall loosening during extension growth of plant cells controlled by the phytohormone auxin. Backbone cleavage of cell wall polysaccharides can be accomplished in vitro by ·OH produced from H2O2 in a Fenton reaction or in a reaction catalyzed by peroxidase supplied with O2 and NADH. Here, we show that coleoptile growth of maize seedlings is accompanied by the release of reactive oxygen intermediates in the cell wall. Auxin promotes release of ${\\mathrm{O}}_{2}^{\\cdot -}$ and subsequent generation of ·OH when inducing elongation growth. Experimental generation of ·OH in the wall causes an increase in wall extensibility in vitro and replaces auxin in inducing growth. Auxin-induced growth can be inhibited by scavengers of ${\\mathrm{O}}_{2}^{\\cdot -}$, H2O2 or ·OH, or inhibitors interfering with the formation of these molecules in the cell wall. These results provide the experimental background for a novel hypothesis on the mechanism of plant cell growth in which ·OH, produced from ${\\mathrm{O}}_{2}^{\\cdot -}$ and H2O2 by cell wall peroxidase, acts as a wall-loosening agent.

Using the tetrazolium salt XTT (Na,3′-[(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzenesulfonic acid hydrate) as a sensitive and physiologically compatible probe for the determination of superoxide (${\\mathrm{O}}_{2}^{\\cdot -}$) production in vivo, we have shown that maize (Zea mays L.) coleoptiles possess the capacity of generating ${\\mathrm{O}}_{2}^{\\cdot -}$ in the apoplastic space. Our results are in agreement with the notion that this activity is localized at the plasma membrane and can be attributed to an ${\\mathrm{O}}_{2}^{\\cdot -}$-synthesizing enzyme with catalytic and kinetic properties similar to that of the NADPH oxidase of mammalian phagocytes, with the important exception that it utilizes NADH instead of NADPH as electron donor. When applied to the apoplastic space, NADH strongly increased the ${\\mathrm{O}}_{2}^{\\cdot -}$-producing activity of coleoptiles. The maize NADH-dependent ${\\mathrm{O}}_{2}^{\\cdot -}$-synthase activity could clearly be differentiated from peroxidase-mediated ${\\mathrm{O}}_{2}^{\\cdot -}$-synthesizing activity by its insensitivity to cyanide and azide, as well as by its much higher affinity to O2. Formation of ${\\mathrm{O}}_{2}^{\\cdot -}$, and concomitantly appearing H2O2, was preferentially localized in the outer epidermis of the coleoptile. The physiological significance of ${\\mathrm{O}}_{2}^{\\cdot -}$ and H2O2 production in relation to the growth-controlling function of the epidermal cell wall is discussed.

Exocytosis in protoplasts from Zea mays L. coleoptiles was studied using patch-clamp techniques. Fusion of individual vesicles with the plasma membrane was monitored as a step increase of the membrane capacitance (Cm). Vesicle fusion was observed as (i) An irreversible step increase in Cm. (ii) Occasionally, irreversible Cm steps were preceded by transient changes in Cm, suggesting that the electrical connection between the vesicle with the plasma membrane opens and closes reversibly before full connection is achieved. (iii) Most frequently, however, stepwise transient changes in Cm did not lead to an irreversible Cm step. Within one patch of membrane capacitance steps due to transient and irreversible fusions were of similar amplitude. This suggests that the exocytosis events do not result from the fusion of vesicles with different sizes but are due to kinetically different states in a fusion process of the same vesicle type. The dwell time histogram of the transient fusion events peaked at about 100 msec. Fusion can be described with a circular three-state model for the fusion process of two fused states and one nonfused state. It predicts that energy input is required to drive the system into a prevailing direction.

The acid-growth response {(AGR) induced by acidic buffer (pH 4) in abraded maize (Zea mays L.) coleoptile segments can be completely inhibited within a few minutes by inhibitors of the haemoprotein function (KCN, Na-azide) or ionophores collapsing the proton gradients across membranes (carbonyl cyanide m-chlorophenyl hydrazone, monensin). These substances also interfere with the acid-mediated increase in cell-wall extensibility measured with a constant-load extensiometer in vivo in turgid or non-turgid segments, but have no effect on the extensibility of the cell walls measured in vitro with frozen/thawed segments. The inhibitors do not cause an alkalinization of the apoplastic solution or a decrease in the osmotic pressure of the cell sap of acid-treated segments. In contrast, inhibitors of ATP synthesis (N, N′-dicyclohexylcarbodiimide, diethylstilbestrol), which arrest auxinmediated growth in a similar way to KCN, have no effect on AGR. Removal of 02 inhibits growth at pH 4 by about 25%; the anoxia-insensitive part of the AGR can be fully inhibited by azide. Diminishing the membrane potential with valinomycin has no effect on AGR. It is concluded that the AGR is controlled by protoplastic functions, possibly localized in the plasma membrane, which are lost when the cells are killed. The isolated cell wall may not represent a sufficient model system for the biochemical mechanism of AGR.

BACKGROUND AND AIMS: Lignin of lignocellulosic residues from biomass for energy can be exploited in sustainable agriculture as plant stimulants. Lignin monomers or their microbial bioproducts are mainly responsible for the plant growth promotion exerted by humic matter in soil. The aim of this work was to verify the humic-like bioactivity of water-soluble lignin isolated from biomass for energy towards plant growth and relate the biostimulation to the lignin molecular structure. METHODS: Two water-soluble lignins isolated from giant reed (AD) and miscanthus (MG) were characterized for molecular composition by ¹H and ³¹P 1D-, ¹³C-¹H 2D-, DOSY-NMR spectroscopy and for conformational structure by size-exclusion chromatography. The effect of different aqueous concentrations of lignin on germination of maize seeds and growth of maize plantlets was assessed in growth-chamber experiments. RESULTS: Both lignins showed humic-like supramolecular structures, but different conformational stability and molecular composition. Their largest bioactivity was revealed at 10 and 50 ppm of lignin organic carbon and both significantly increased length of radicles, lateral seminal roots, and coleoptiles of maize seedlings, as well as total shoot and root dry weights and root length of maize plantlets. However, differences in AD and MG bioactivity were attributed to their conformational stabilities and content of amphiphilic molecules, which may control both the adhesion to plant roots and the release of bioactive molecules upon interactions with plant-exuded organic acids. CONCLUSIONS: The humic-like bioactivity of water-soluble lignins indicated that lignocellulosic residues from energy crops may be profitably recycled in agriculture as effective plant growth promoters, thereby increasing the economic and environmental sustainability of energy production from non-food biomasses.