Arabidopsis vascular complexity and connectivity controls PIN-FORMED1 dynamics and lateral vein patterning during embryogenesis.

Arabidopsis VASCULATURE COMPLEXITY AND CONNECTIVITY (VCC) is a plant-specific transmembrane protein that controls the development of veins in cotyledons. Here, we show that the expression and localization of the auxin efflux carrier PIN-FORMED1 (PIN1) is altered in vcc developing cotyledons and that overexpression of PIN1-GFP partially rescues vascular defects of vcc in a dosage-dependent manner. Genetic analyses suggest that VCC and PINOID (PID), a kinase that regulates PIN1 polarity, are both required for PIN1-mediated control of vasculature development. VCC expression is upregulated by auxin, likely as part of a positive feedback loop for the progression of vascular development. VCC and PIN1 localized to the plasma membrane in pre-procambial cells but were actively redirected to vacuoles in procambial cells for degradation. In the vcc mutant, PIN1 failed to properly polarize in pre-procambial cells during the formation of basal strands, and instead, it was prematurely degraded in vacuoles. VCC plays a role in the localization and stability of PIN1, which is crucial for the transition of pre-procambial cells into procambial cells that are involved in the formation of basal lateral strands in embryonic cotyledons.

Localization and Dynamics of the Methionine Sulfoxide Reductases MsrB1 and MsrB2 in Beech Seeds.

Beech seeds are produced irregularly, and there is a need for long-term storage of these seeds for forest management practices. Accumulated reactive oxygen species broadly oxidize molecules, including amino acids, such as methionine, thereby contributing to decreased seed viability. Methionine oxidation can be reversed by the activity of methionine sulfoxide reductases (Msrs), which are enzymes involved in the regulation of many developmental processes and stress responses. Two types of Msrs, MsrB1 and MsrB2, were investigated in beech seeds to determine their abundance and localization. MsrB1 and MsrB2 were detected in the cortical cells and the outer area of the vascular cylinder of the embryonic axes as well as in the epidermis and parenchyma cells of cotyledons. The abundances of MsrB1 and MsrB2 decreased during long-term storage. Ultrastructural analyses have demonstrated the accumulation of these proteins in protein storage vacuoles and in the cytoplasm, especially in close proximity to the cell membrane. In silico predictions of possible Msr interactions supported our findings. In this study, we investigate the contribution of MsrB1 and MsrB2 locations in the regulation of seed viability and suggest that MsrB2 is linked with the longevity of beech seeds via association with proper utilization of storage material.

Deep Imaging Analysis in VISUAL Reveals the Role of YABBY Genes in Vascular Stem Cell Fate Determination.

Stem cells undergo cell division and differentiation to ensure organized tissue development. Because plant cells are immobile, plant stem cells ought to decide their cell fate prior to differentiation, to locate specialized cells in the correct position. In this study, based on a chemical screen, we isolated a novel secondary cell wall indicator BF-170, which binds to lignin and can be used to image in vitro and in situ xylem development. Use of BF-170 to observe the vascular differentiation pattern in the in vitro vascular cell induction system, VISUAL, revealed that adaxial mesophyll cells of cotyledons predominantly generate ectopic xylem cells. Moreover, phloem cells are abundantly produced on the abaxial layer, suggesting the involvement of leaf adaxial-abaxial polarity in determining vascular cell fate. Analysis of abaxial polarity mutants highlighted the role of YAB3, an abaxial cell fate regulator, in suppressing xylem and promoting phloem differentiation on the abaxial domains in VISUAL. Furthermore, YABBY family genes affected in vivo vascular development during the secondary growth. Our results denoted the possibility that such mediators of spatial information contribute to correctly determine the cell fate of vascular stem cells, to conserve the vascular pattern of land plants.

Simultaneous visualization of callose deposition and plasma membrane for live-cell imaging in plants.

KEY MESSAGE: The appropriate combination of fluorescent probes enabled the simultaneous visualization of callose deposition and plasma membrane in living Arabidopsis and can be useful for the cell biological study of papilla formation in plants. Localized callose deposition at the site of fungal infection is a central part of papilla formation, which creates a barrier between the host plasma membrane and the cell wall and plays an important role in preventing the penetration of fungal hyphae into the host cells. Using chitin-induced callose deposition as a model system, we examined suitable conditions for the simultaneous visualization of callose deposition and plasma membrane dynamics in living Arabidopsis cotyledons. We found that aniline blue fluorochrome (ABF) for callose staining selectively interferes with FM dyes for membrane visualization depending on the structure of the latter compounds and the proper combination of these fluorescent dyes and staining conditions is a key for successful live-cell imaging. The established conditions enabled the live-cell imaging of chitin-induced callose deposition and host membrane systems. The established system/conditions would also be useful for the cell biological studies on the localized callose deposition in other stress/development-associated processes. The finding that the slight difference in the structure of FM dyes affects the interaction with another fluorescent dye, ABF, would also give useful suggestions for the studies where multiple fluorescent dyes are utilized for live-cell imaging.

Transcription Factors VND1-VND3 Contribute to Cotyledon Xylem Vessel Formation.

Arabidopsis (Arabidopsis thaliana) VASCULAR-RELATED NAC-DOMAIN1 (VND1) to VND7 encode a group of NAC domain transcription factors that function as master regulators of xylem vessel element differentiation. These transcription factors activate the transcription of genes required for secondary cell wall formation and programmed cell death, key events in xylem vessel element differentiation. Because constitutive overexpression of VND6 and VND7 induces ectopic xylem vessel element differentiation, functional studies of VND proteins have largely focused on these two proteins. Here, we report the roles of VND1, VND2, and VND3 in xylem vessel formation in cotyledons. Using our newly established in vitro system in which excised Arabidopsis cotyledons are stimulated to undergo xylem cell differentiation by cytokinin, auxin, and brassinosteroid treatment, we found that ectopic xylem vessel element differentiation required VND1, VND2, and VND3 but not VND6 or VND7. The importance of VND1, VND2, and VND3 also was indicated in vivo; in the vnd1 vnd2 vnd3 seedlings, xylem vessel element differentiation of secondary veins in cotyledons was inhibited under dark conditions. Furthermore, the light responsiveness of VND gene expression was disturbed in the vnd1 vnd2 vnd3 mutant, and vnd1 vnd2 vnd3 failed to recover lateral root development in response to the change of light conditions. These findings suggest that VND1 to VND3 have specific molecular functions, possibly linking light conditions to xylem vessel formation, during seedling development.

Commelinid Monocotyledon Lignins Are Acylated by p-Coumarate.

Commelinid monocotyledons are a monophyletic clade differentiated from other monocotyledons by the presence of cell wall-bound ferulate and p-coumarate. The Poaceae, or grass family, is a member of this group, and most of the p-coumarate in the cell walls of this family acylates lignin. Here, we isolated and examined lignified cell wall preparations from 10 species of commelinid monocotyledons from nine families other than Poaceae, including species from all four commelinid monocotyledon orders (Poales, Zingiberales, Commelinales, and Arecales). We showed that, as in the Poaceae, lignin-linked p-coumarate occurs exclusively on the hydroxyl group on the γ-carbon of lignin unit side chains, mostly on syringyl units. Although the mechanism of acylation has not been studied directly in these species, it is likely to be similar to that in the Poaceae and involve BAHD acyl-coenzyme A:monolignol transferases.

The Microtubule-Associated Protein IQ67 DOMAIN5 Modulates Microtubule Dynamics and Pavement Cell Shape.

The dynamic arrangement of cortical microtubules (MTs) plays a pivotal role in controlling cell growth and shape formation in plants, but the mechanisms by which cortical MTs are organized to regulate these processes are not well characterized. In particular, the dynamic behavior of cortical MTs is critical for their spatial organization, yet the molecular mechanisms controlling MT dynamics remain poorly understood. In this study, we used the puzzle piece-shaped pavement cells of Arabidopsis (Arabidopsis thaliana) leaves as a model system in which to study cortical MT organization. We isolated an ethyl methanesulfonate mutant with reduced interdigitation of pavement cells in cotyledons. This line carried a mutation in IQ67 DOMAIN5 (IQD5), which encodes a member of the plant-specific IQ motif protein family. Live-cell imaging and biochemical analyses demonstrated that IQD5 binds to MTs and promotes MT assembly. MT-depolymerizing drug treatment and in vivo MT dynamics assays suggested that IQD5 functions to stabilize MTs. Hence, our findings provide genetic, cell biological, and biochemical evidence that IQD5 regulates MT dynamics that affect MT organization and subsequent cell shape formation.

New Insight on Water Status in Germinating Brassica napus Seeds in Relation to Priming-Improved Germination.

Seed priming is a pre-sowing method successfully used to improve seed germination. Since water plays a crucial role in germination, the aim of this study was to investigate the relationship between better germination performances of osmoprimed Brassica napus seeds and seed water status during germination. To achieve this goal, a combination of different kinds of approaches was used, including nuclear magnetic resonance (NMR) spectroscopy, TEM, and SEM as well as semi-quantitative PCR (semi-qPCR). The results of this study showed that osmopriming enhanced the kinetics of water uptake and the total amount of absorbed water during both the early imbibition stage and in the later phases of seed germination. The spin⁻spin relaxation time (T2) measurement suggests that osmopriming causes faster water penetration into the seed and more efficient tissue hydration. Moreover, factors potentially affecting water relations in germinating primed seeds were also identified. It was shown that osmopriming (i) changes the microstructural features of the seed coat, e.g., leads to the formation of microcracks, (ii) alters the internal structure of the seed by the induction of additional void spaces in the seed, (iii) increases cotyledons cells vacuolization, and (iv) modifies the expression pattern of aquaporin genes.

DEFECTIVE KERNEL 1 promotes and maintains plant epidermal differentiation.

During plant epidermal development, many cell types are generated from protodermal cells, a process requiring complex co-ordination of cell division, growth, endoreduplication and the acquisition of differentiated cellular morphologies. Here we show that the Arabidopsis phytocalpain DEFECTIVE KERNEL 1 (DEK1) promotes the differentiated epidermal state. Plants with reduced DEK1 activity produce cotyledon epidermis with protodermal characteristics, despite showing normal growth and endoreduplication. Furthermore, in non-embryonic tissues (true leaves, sepals), DEK1 is required for epidermis differentiation maintenance. We show that the HD-ZIP IV family of epidermis-specific differentiation-promoting transcription factors are key, albeit indirect, targets of DEK1 activity. We propose a model in which DEK1 influences HD-ZIP IV gene expression, and thus epidermis differentiation, by promoting cell adhesion and communication in the epidermis.

Mitogen-activated protein kinases concentrate in the vicinity of chromosomes and may regulate directly cellular patterning in Vicia faba embryos.

MAIN CONCLUSION: Mitogen-activated protein kinases seem to mark genes which are set up to be activated in daughter cells and thus they may play a direct role in cellular patterning during embryogenesis. Embryonic patterning starts very early and after the first division of zygote different genes are expressed in apical and basal cells. However, there is an ongoing debate about the way these different transcription patterns are established during embryogenesis. The presented data indicate that mitogen-activated protein kinases (MAPKs) concentrate in the vicinity of chromosomes and form visible foci there. Cells in the apical and basal regions differ in number of foci observed during the metaphase which suggests that cellular patterning may be determined by activation of diverse MAPK-dependent genes. Different number of foci in each group of separating chromatids and the specified direction of these mitoses in apical-basal axis indicate that the unilateral auxin accumulation in a single cell may regulate the number of foci in each group of chromatids. Thus, we put forward a hypothesis that MAPKs localized in the vicinity of chromosomes during mitosis mark those genes which are set up to be activated in daughter cells after division. It implies that the chromosomal localization of MAPKs may be one of the mechanisms involved in establishment of cellular patterns in some plant species.

Differential Growth in Periclinal and Anticlinal Walls during Lobe Formation in Arabidopsis Cotyledon Pavement Cells.

Lobe development in the epidermal pavement cells of Arabidopsis thaliana cotyledons and leaves is thought to take place via tip-like growth on the concave side of lobes driven by localized concentrations of actin filaments and associated proteins, with a predicted role for cortical microtubules in establishing the direction of restricted growth at the convex side. We used homologous landmarks fixed to the outer walls of pavement cells and thin-plate spline analysis to demonstrate that lobes form by differential growth of both the anticlinal and periclinal walls. Most lobes formed within the first 24 h of the cotyledons unfurling, during the period of rapid cell expansion. Cortical microtubules adjacent to the periclinal wall were persistently enriched at the convex side of lobes during development where growth was anisotropic and were less concentrated or absent at the concave side where growth was promoted. Alternating microtubule-enriched and microtubule-free zones at the periclinal wall in neighboring cells predicted sites of new lobes. There was no particular arrangement of cortical actin filaments that could predict where lobes would form. However, drug studies demonstrate that both filamentous actin and microtubules are required for lobe formation.

Exogenous Cellulase Switches Cell Interdigitation to Cell Elongation in an RIC1-dependent Manner in Arabidopsis thaliana Cotyledon Pavement Cells.

Pavement cells in cotyledons and true leaves exhibit a jigsaw puzzle-like morphology in most dicotyledonous plants. Among the molecular mechanisms mediating cell morphogenesis, two antagonistic Rho-like GTPases regulate local cell outgrowth via cytoskeletal rearrangements. Analyses of several cell wall-related mutants suggest the importance of cell wall mechanics in the formation of interdigitated patterns. However, how these factors are integrated is unknown. In this study, we observed that the application of exogenous cellulase to hydroponically grown Arabidopsis thaliana cotyledons switched the interdigitation of pavement cells to the production of smoothly elongated cells. The cellulase-induced inhibition of cell interdigitation was not observed in a RIC1 knockout mutant. This gene encodes a Rho-like GTPase-interacting protein important for localized cell growth suppression via microtubule bundling on concave cell interfaces. Additionally, to characterize pavement cell morphologies, we developed a mathematical model that considers the balance between cell and cell wall growth, restricted global cell growth orientation, and regulation of local cell outgrowth mediated by a Rho-like GTPase-cytoskeleton system. Our computational simulations fully support our experimental observations, and suggest that interdigitated patterns form because of mechanical buckling in the absence of Rho-like GTPase-dependent regulation of local cell outgrowth. Our model clarifies the cell wall mechanics influencing pavement cell morphogenesis.

Novel Insights into the Organization of Laticifer Cells: A Cell Comprising a Unified Whole System.

Laticifer cells are specialized plant cells that synthesize and accumulate latex. Studies on laticifers have lagged behind in recent years, and data regarding the functional role of laticifers and their fitness benefit still remain elusive. Laticifer differentiation and its impact on plant growth and development also remain to be investigated. Here, cellular, molecular, and genetic tools were developed to examine the distribution, differentiation, ontogeny, and other characteristic features, as well as the potential developmental role of laticifer cells in the latex-bearing plant Euphorbia lathyris. The organization of the laticiferous system within the E. lathyris plant body is reported, emerging as a single elongated and branched coenocytic cell, constituting the largest cell type existing in plants. We also report the ontogeny and organization of laticifer cells in the embryo and the identification of a laticifer-associated gene expression pattern. Moreover, the identification of laticifer- and latex-deficient mutants (pil mutants) allowed for the identification of distinct loci regulating laticifer differentiation, growth, and metabolic activity. Additionally, pil mutants revealed that laticifer cells appear nonessential for plant growth and development, thus pointing toward their importance, instead, for specific ecophysiological adaptations of latex-bearing plants in natural environments.

Differential Cellular Control by Cotyledon-Derived Phytohormones Involved in Graft Reunion of Arabidopsis Hypocotyls.

When wounding or grafting interrupts the original connection of plant tissue, cell proliferation is induced and the divided tissue is reunited. Previous studies suggested that gibberellin derived from the cotyledon is required for tissue reunion in cucumber and tomato incised hypocotyls, and tissue reunion of Arabidopsis incised flowering stems is controlled by auxin. Differences in the hormone requirements of the tissue reunion process between Arabidopsis and cucumber might be due to differences in organs or species. In this study, we performed morphological and gene expression analyses of graft union in Arabidopsis hypocotyl. We found that removal of the cotyledon and treatment of the cotyledon with the auxin transport inhibitor triiodobenzoic acid (TIBA) suppressed cell proliferation of vascular tissue during graft union formation. These treatments also suppressed expression of IAA5, ANAC071, ANAC096 and CYCB1;1. ANAC071 is involved in the tissue reunion process. The anac071 anac096 double mutant suppressed cell proliferation more so than either of the single mutants. On the other hand, paclobutrazol treatment or deficiency of gibberellin biosynthesis genes suppressed expansion of cortex cells, and exogenous gibberellin treatment or rga/gai mutations that lack the negative regulator of gibberellin reversed this inhibition. The up-regulation of the key gibberellin biosynthesis gene GA20ox1 during graft union formation was prevented by cotyledon removal or TIBA treatment. These data suggest that auxin regulates cell proliferation of vascular tissue and expansion of cortex cells by promoting gibberellin biosynthesis during graft attachment. We hypothesize that the cotyledon-derived phytohormones are essential for graft reunion of the hypocotyl, processed in a cell type-specific manner, in Arabidopsis.

Arabidopsis homeodomain-leucine zipper IV proteins promote stomatal development and ectopically induce stomata beyond the epidermis.

The shoot epidermis of land plants serves as a crucial interface between plants and the atmosphere: pavement cells protect plants from desiccation and other environmental stresses, while stomata facilitate gas exchange and transpiration. Advances have been made in our understanding of stomatal patterning and differentiation, and a set of 'master regulatory' transcription factors of stomatal development have been identified. However, they are limited to specifying stomatal differentiation within the epidermis. Here, we report the identification of an Arabidopsis homeodomain-leucine zipper IV (HD-ZIP IV) protein, HOMEODOMAIN GLABROUS2 (HDG2), as a key epidermal component promoting stomatal differentiation. HDG2 is highly enriched in meristemoids, which are transient-amplifying populations of stomatal-cell lineages. Ectopic expression of HDG2 confers differentiation of stomata in internal mesophyll tissues and occasional multiple epidermal layers. Conversely, a loss-of-function hdg2 mutation delays stomatal differentiation and, rarely but consistently, results in aberrant stomata. A closely related HD-ZIP IV gene, Arabidopsis thaliana MERISTEM LAYER1 (AtML1), shares overlapping function with HDG2: AtML1 overexpression also triggers ectopic stomatal differentiation in the mesophyll layer and atml1 mutation enhances the stomatal differentiation defects of hdg2. Consistently, HDG2 and AtML1 bind the same DNA elements, and activate transcription in yeast. Furthermore, HDG2 transactivates expression of genes that regulate stomatal development in planta. Our study highlights the similarities and uniqueness of these two HD-ZIP IV genes in the specification of protodermal identity and stomatal differentiation beyond predetermined tissue layers.

A mutation of casein kinase 2 α4 subunit affects multiple developmental processes in Arabidopsis.

KEY MESSAGE: Arabidopsis CK2 α4 subunit regulates the primary root and hypocotyl elongation, lateral root formation, cotyledon expansion, rosette leaf initiation and growth, flowering, and anthocyanin biosynthesis. Casein kinase 2 (CK2) is a conserved tetrameric kinase composed of two α and two ß subunits. The inhibition of CK2 activity usually results in severe developmental deficiency. Four genes (CKA1-CKA4) encode CK2 α subunit in Arabidopsis. Single mutations of CKA1, CKA2, and CKA3 do not affect the normal growth of Arabidopsis, while the cka1 cka2 cka3 triple mutants are defective in cotyledon and hypocotyl growth, lateral root development, and flowering. The inhibition of CKA4 expression in cka1 cka2 cka3 background further reduces the number of lateral roots and delays the flowering time. Here, we report the characterization of a novel knockout mutant of CKA4, which exhibits various developmental defects including reduced primary root and hypocotyl elongation, increased lateral root density, delayed cotyledon expansion, retarded rosette leaf initiation and growth, and late flowering. The examination of the cellular basis for abnormal root development of this mutant revealed reduced root meristem cells with enhanced RETINOBLASTOMA-RELATED (RBR) expression that promotes cell differentiation in root meristem. Moreover, this cka4-2 mutant accumulates higher anthocyanin in the aerial part and shows an increased expression of anthocyanin biosynthetic genes, suggesting a novel role of CK2 in modulating anthocyanin biosynthesis. In addition, the complementation test using primary root elongation assay as a sample confirms that the changed phenotypes of this cka4-2 mutant are due to the lack of CKA4. Taken together, this study reveals an essential role of CK2 α4 subunit in multiple developmental processes in Arabidopsis.

Agrobacterium-mediated transformation of the ß-subunit gene in 7S globulin protein in soybean using RNAi technology.

The objective of this study was to use RNA interference (RNAi) to improve protein quality and decrease anti-nutritional effects in soybean. Agrobacterium tumefaciens-mediated transformation was conducted using RNAi and an expression vector containing the 7S globulin ß-subunit gene. The BAR gene was used as the selective marker and cotyledonary nodes of soybean genotype Jinong 27 were chosen as explant material. Regenerated plants were detected by molecular biology techniques. Transformation of the ß-subunit gene in the 7S protein was detected by PCR, Southern blot, and q-PCR. Positive plants (10 T0, and 6 T1, and 13 T2) were tested by PCR. Hybridization bands were detected by Southern blot analysis in two of the T1 transgenic plants. RNAi expression vectors containing the soybean 7S protein ß-subunit gene were successfully integrated into the genome of transgenic plants. qRT-PCR analysis in soybean seeds showed a clear decrease in expression of the soybean ß-subunit gene. The level of 7S protein ß-subunit expression in transgenic plants decreased by 77.5% as compared to that of the wild-type plants. This study has established a basis for the application of RNAi to improve the anti-nutritional effects of soybean.

Differential transcriptional networks associated with key phases of ingrowth wall construction in trans-differentiating epidermal transfer cells of Vicia faba cotyledons.

BACKGROUND: Transfer cells are characterized by intricate ingrowth walls, comprising an uniform wall upon which wall ingrowths are deposited. The ingrowth wall forms a scaffold to support an amplified plasma membrane surface area enriched in membrane transporters that collectively confers transfer cells with an enhanced capacity for membrane transport at bottlenecks for apo-/symplasmic exchange of nutrients. However, the underlying molecular mechanisms regulating polarized construction of the ingrowth wall and membrane transporter profile are poorly understood. RESULTS: An RNAseq study of an inducible epidermal transfer cell system in cultured Vicia faba cotyledons identified transfer cell specific transcriptomes associated with uniform wall and wall ingrowth deposition. All functional groups of genes examined were expressed before and following transition to a transfer cell fate. What changed were the isoform profiles of expressed genes within functional groups. Genes encoding ethylene and Ca(2+) signal generation and transduction pathways were enriched during uniform wall construction. Auxin-and reactive oxygen species-related genes dominated during wall ingrowth formation and ABA genes were evenly expressed across ingrowth wall construction. Expression of genes encoding kinesins, formins and villins was consistent with reorganization of cytoskeletal components. Uniform wall and wall ingrowth specific expression of exocyst complex components and SNAREs suggested specific patterns of exocytosis while dynamin mediated endocytotic activity was consistent with establishing wall ingrowth loci. Key regulatory genes of biosynthetic pathways for sphingolipids and sterols were expressed across ingrowth wall construction. Transfer cell specific expression of cellulose synthases was absent. Rather xyloglucan, xylan and pectin biosynthetic genes were selectively expressed during uniform wall construction. More striking was expression of genes encoding enzymes for re-modelling/degradation of cellulose, xyloglucans, pectins and callose. Extensins dominated the cohort of expressed wall structural proteins and particularly so across wall ingrowth development. Ion transporters were selectively expressed throughout ingrowth wall development along with organic nitrogen transporters and a large group of ABC transporters. Sugar transporters were less represented. CONCLUSIONS: Pathways regulating signalling and intracellular organization were fine tuned whilst cell wall construction and membrane transporter profiles were altered substantially upon transiting to a transfer cell fate. Each phase of ingrowth wall construction was linked with unique cohorts of expressed genes.

Inducible knockdown of MONOGALACTOSYLDIACYLGLYCEROL SYNTHASE1 reveals roles of galactolipids in organelle differentiation in Arabidopsis cotyledons.

Monogalactosyldiacylglycerol (MGDG) is the major lipid constituent of thylakoid membranes and is essential for chloroplast biogenesis in plants. In Arabidopsis (Arabidopsis thaliana), MGDG is predominantly synthesized by inner envelope-localized MONOGALACTOSYLDIACYLGLYCEROL SYNTHASE1 (MGD1); its knockout causes albino seedlings. Because of the lethal phenotype of the null MGD1 mutant, functional details of MGDG synthesis at seedling development have remained elusive. In this study, we used an inducible gene-suppression system to investigate the impact of MGDG synthesis on cotyledon development. We created transgenic Arabidopsis lines that express an artificial microRNA targeting MGD1 (amiR-MGD1) under the control of a dexamethasone-inducible promoter. The induction of amiR-MGD1 resulted in up to 75% suppression of MGD1 expression, although the resulting phenotypes related to chloroplast development were diverse, even within a line. The strong MGD1 suppression by continuous dexamethasone treatment caused substantial decreases in galactolipid content in cotyledons, leading to severe defects in the formation of thylakoid membranes and impaired photosynthetic electron transport. Time-course analyses of the MGD1 suppression during seedling germination revealed that MGDG synthesis at the very early germination stage is particularly important for chloroplast biogenesis. The MGD1 suppression down-regulated genes associated with the photorespiratory pathway in peroxisomes and mitochondria as well as those responsible for photosynthesis in chloroplasts and caused high expression of genes for the glyoxylate cycle. MGD1 function may link galactolipid synthesis with the coordinated transcriptional regulation of chloroplasts and other organelles during cotyledon greening.

Calcium-dependent depletion zones in the cortical microtubule array coincide with sites of, but do not regulate, wall ingrowth papillae deposition in epidermal transfer cells.

Trans-differentiation to a transfer-cell morphology is characterized by the localized deposition of wall ingrowth papillae that protrude into the cytosol. Whether the cortical microtubule array directs wall ingrowth papillae formation was investigated using a Vicia faba cotyledon culture system in which their adaxial epidermal cells were spontaneously induced to trans-differentiate to transfer cells. During deposition of wall ingrowth papillae, the aligned cortical microtubule arrays in precursor epidermal cells were reorganized into a randomized array characterized by circular depletion zones. Concurrence of the temporal appearance, spatial pattern, and size of depletion zones and wall ingrowth papillae was consistent with each papilla occupying a depletion zone. Surprisingly, microtubules appeared not to regulate construction of wall ingrowth papillae, as neither depolymerization nor stabilization of cortical microtubules changed their deposition pattern or morphology. Moreover, the size and spatial pattern of depletion zones was unaltered when the formation of wall ingrowth papillae was blocked by inhibiting cellulose biosynthesis. In contrast, the depletion zones were absent when the cytosolic calcium plumes, responsible for directing wall ingrowth papillae formation, were blocked or dissipated. Thus, we conclude that the depletion zones within the cortical microtubule array result from localized depolymerization of microtubules initiated by elevated cytosolic Ca(2+) levels at loci where wall ingrowth papillae are deposited. The physiological significance of the depletion zones as a mechanism to accommodate the construction of wall ingrowth papillae without compromising maintenance of the plasma membrane-microtubule inter-relationship is discussed.