A survey of cellulose microfibril patterns in dividing,expanding, and differentiating cells of Arabidopsis thaliana

Cellulose microfibrils are critical for plant cell specialization and function. Recent advances in live cell imaging of fluorescently tagged cellulose synthases to track cellulose synthesis have greatly advanced our understanding of cellulose biosynthesis. Nevertheless, cellulose deposition patterns remain poorly described in many cell types, including those in the process of division or differentiation. In this study, we used field emission scanning electron microscopy analysis of cryo-planed tissues to determine the arrangement of cellulose microfibrils in various faces of cells undergoing cytokinesis or specialized development, including cell types in which cellulose cannot be imaged by conventional approaches. In dividing cells, we detected microfibrillar meshworks in the cell plates, consistent with the concentration at the cell plate of cellulose synthase complexes, as detected by fluorescently tagged CesA6. We also observed a loss of parallel cellulose microfibril orientation in walls of the mother cell during cytokinesis, which corresponded with the loss of fluorescently tagged cellulose synthase complexes from these surfaces. In recently formed guard cells, microfibrils were randomly organized and only formed a highly ordered circumferential pattern after pore formation. In pit fields, cellulose microfibrils were arranged in circular patterns around plasmodesmata. Microfibrils were random in most cotyledon cells except the epidermis and were parallel to the growth axis in trichomes. Deposition of cellulose microfibrils was spatially delineated in metaxylem and protoxylem cells of the inflorescence stem, supporting recent studies on microtubule exclusion mechanisms.     

Root hair-specific disruption of cellulose and xyloglucan in <Emphasis Type="Italic">AtCSLD3</Emphasis> mutants,and factors affecting the post-rupture resumption of mutant root hair growth

The glycosyl transferase encoded by the cellulose synthase-like gene CSLD3/KJK/RHD7 (At3g03050) is required for cell wall integrity during root hair formation in Arabidopsis thaliana but it remains unclear whether it contributes to the synthesis of cellulose or hemicellulose. We identified two new alleles, root hair-defective (rhd) 7-1 and rhd7-4, which affect the C-terminal end of the encoded protein. Like root hairs in the previously characterized kjk-2 putative null mutant, rhd7-1 and rhd7-4 hairs rupture before tip growth but, depending on the growth medium and temperature, hairs are able to survive rupture and initiate tip growth, indicating that these alleles retain some function. At 21°C, the rhd7 tip-growing root hairs continued to rupture but at 5oC, rupture was inhibited, resulting in long, wild type-like root hairs. At both temperatures, the expression of another root hair-specific CSLD gene, CSLD2, was increased in the rhd7-4 mutant but reduced in the kjk-2 mutant, suggesting that CSLD2 expression is CSLD3-dependent, and that CSLD2 could partially compensate for CSLD3 defects to prevent rupture at 5°C. Using a fluorescent brightener (FB 28) to detect cell wall (1 → 4)-β-glucans (primarily cellulose) and CCRC-M1 antibody to detect fucosylated xyloglucans revealed a patchy distribution of both in the mutant root hair cell walls. Cell wall thickness varied, and immunogold electron microscopy indicated that xyloglucan distribution was altered throughout the root hair cell walls. These cell wall defects indicate that CSLD3 is required for the normal organization of both cellulose and xyloglucan in root hair cell walls.     

Trafficking of the myrosinase‐associated protein GLL23 requires NUC/MVP1/GOLD36/ERMO3 and the p24 protein CYB

Proteins detrimental to endoplasmic reticulum (ER) morphology need to be efficiently exported. Here, we identify two mechanisms that control trafficking of Arabidopsis thalianaGLL23, a 43 kDa GDSL‐like lipase implicated in glucosinolate metabolism through its association with the β‐glucosidase myrosinase. Using immunofluorescence, we identified two mutants that showed aberrant accumulation of GLL23: large perinuclear ER aggregates in the nuclear cage (nuc) mutant; and small compartments contiguous with the peripheral ER in the cytoplasmic bodies (cyb) mutant. Live imaging of fluorescently tagged GLL23 confirmed its presence in the nuc and cyb compartments, but lack of fluorescent signals in the wild‐type plants suggested that GLL23 is normally post‐translationally modified for ER export. NUC encodes the MVP1/GOLD36/ERMO3 myrosinase‐associated protein, previously shown to have vacuolar distribution. CYB is an ER and Golgi‐localized p24 type I membrane protein component of coat protein complex (COP) vesicles, animal and yeast homologues of which are known to be involved in selective cargo sorting for ER–Golgi export. Without NUC, GLL23 accumulates in the ER this situation suggests that NUC is in fact active in the ER. Without CYB, both GLL23 and NUC were found to accumulate in cyb compartments, consistent with a role for NUC in GLL23 processing and indicated that GLL23 is the likely sorting target of the CYB p24 protein.     

The Microtubule Plus-End Tracking Protein ARMADILLO-REPEAT KINESIN1 Promotes Microtubule Catastrophe in Arabidopsis

Microtubule dynamics are critically important for plant cell development. Here, we show that Arabidopsis thaliana ARMADILLO-REPEAT KINESIN1 (ARK1) plays a key role in root hair tip growth by promoting microtubule catastrophe events. This destabilizing activity appears to maintain adequate free tubulin concentrations in order to permit rapid microtubule growth, which in turn is correlated with uniform tip growth. Microtubules in ark1-1 root hairs exhibited reduced catastrophe frequency and slower growth velocities, both of which were restored by low concentrations of the microtubule-destabilizing drug oryzalin. An ARK1-GFP (green fluorescent protein) fusion protein expressed under its endogenous promoter localized to growing microtubule plus ends and rescued the ark1-1 root hair phenotype. Transient overexpression of ARK1-RFP (red fluorescent protein) increased microtubule catastrophe frequency. ARK1-fusion protein constructs lacking the N-terminal motor domain still labeled microtubules, suggesting the existence of a second microtubule binding domain at the C terminus of ARK1. ARK1-GFP was broadly expressed in seedlings, but mutant phenotypes were restricted to root hairs, indicating that ARK1’s function is redundant in cells other than those forming root hairs.     

Nanoscale and geometric influences on the microtubule cytoskeleton in plants: thinking inside and outside the box

The dynamic microtubule (MT) cytoskeleton found in the cell cortex of plants drives cell expansion via cell wall modifications. In the last decade, live cell imaging studies employing green fluorescent protein have helped unravel the mechanisms behind how cells arrange cortical MTs into complex arrays and shape cell expansion. In this review, we explore the reverse scenario: how cell geometry and organelles influence and constrain the organization and behavior of cortical MTs. This newly emerging principle explains how cells perceive local nanoscale structural input from MT-organizing centers, such as the nucleus, endomembranes, and cell edges, and translate this into global cell-wide order via MT self-organization. Studies primarily using the model plant Arabidopsis thaliana and tobacco BY-2 suspension cultures have broadened our understanding of how cells form not only elegant parallel arrays but also more complex MT configurations, including the prominent MT bundles found in preprophase bands, leaf epidermal cells, and developing xylem.     

Red-listed lichens in Sweden: habitats, threats, protection, and indicator value in boreal coniferous forests

The three major threats to lichens in Sweden; forestry, air pollution, and structural changes in the agricultural landscape, are outlined. Forestry is the most serious. The majority of the red-listed lichens in forests are found in productive forests with a continuity in tree growth, but nearly 70% of the protected area in Sweden consists of alpine landscapes. Examples of important habitats with red-listed lichens are described, e.g. Alnus glutinosa marshes, alvar-vegetation, broad-leaved deciduous forests and different Picea abies forests. The use of selected indicator species of lichens and macrofungi (mainly red-listed species) in boreal coniferous forests provides information on continuity in tree growth of an area, and on earlier influences of forestry. Examples of indicator species for different types of forest are presented and discussed. In spite of the Swedish right of common accesso, collecting of lichens is forbidden without permission from the land-owner. Collecting in protected areas is allowed only with a permit obtained from the authorities.     

Tree diversity drives abundance and spatiotemporal β‐diversity of true bugs (Heteroptera)

1. Spatiotemporal patterns of canopy true bug diversity in forests of different tree species diversity have not yet been disentangled, although plant diversity has been shown to strongly impact the diversity and distribution of many insect communities. 2. Here we compare species richness of canopy true bugs across a tree diversity gradient ranging from simple beech to mixed forest stands. We analyse changes in community composition by additive partitioning of species diversity, for communities on various tree species, as well as for communities dwelling on beech alone. 3. Total species richness (γ‐diversity) and α‐diversity, and abundance of true bugs increased across the tree diversity gradient, while diversity changes were mediated by increased true bug abundance in the highly diverse forest stands. The same pattern was found for γ‐diversity in most functional guilds (e.g. forest specialists, herbivores, predators). Temporal and even more, spatial turnover (β‐diversity) among trees was closely related to tree diversity and accounted for ～90% of total γ‐diversity. 4. Results for beech alone were similar, but species turnover could not be related to the tree diversity gradient, and monthly turnover was higher compared to turnover among trees. 5. Our findings support the hypothesis that with increasing tree diversity and thereby increasing habitat heterogeneity, enhanced resource availability supports a greater number of individuals and species of true bugs. Tree species identity and the dissimilarity of true bug communities from tree to tree determine community patterns. 6. In conclusion, understanding diversity and distribution of insect communities in deciduous forests needs a perspective on patterns of spatiotemporal turnover. Heterogeneity among sites, tree species, as well as tree individuals contributed greatly to overall bug diversity.     

New techniques enable comparative analysis of microtubule orientation, wall texture, and growth rate in intact roots of Arabidopsis

This article explores root epidermal cell elongation and its dependence on two structural elements of cells, cortical microtubules and cellulose microfibrils. The recent identification of Arabidopsis morphology mutants with putative cell wall or cytoskeletal defects demands a procedure for examining and comparing wall architecture and microtubule organization patterns in this species. We developed methods to examine cellulose microfibrils by field emission scanning electron microscopy and microtubules by immunofluorescence in essentially intact roots. We were able to compare cellulose microfibril and microtubule alignment patterns at equivalent stages of cell expansion. Field emission scanning electron microscopy revealed that Arabidopsis root epidermal cells have typical dicot primary cell wall structure with prominent transverse cellulose microfibrils embedded in pectic substances. Our analysis showed that microtubules and microfibrils have similar orientation only during the initial phase of elongation growth. Microtubule patterns deviate from a predominantly transverse orientation while cells are still expanding, whereas cellulose microfibrils remain transverse until well after expansion finishes. We also observed microtubule-microfibril alignment discord before cells enter their elongation phase. This study and the new technology it presents provide a starting point for further investigations on the physical properties of cell walls and their mechanisms of assembly.