Nanosized extracellular vesicles released by Neurospora crassa hyphae.

Extracellular vesicles (EVs) are nanosized structures containing proteins, lipids, and nucleic acids, released by living cells to the surrounding medium. EVs participate in diverse processes, such as intercellular communication, virulence, and disease. In pathogenic fungi, EVs carry enzymes that allow them to invade the host or undergo environmental adaptation successfully. In Neurospora crassa, a non-pathogenic filamentous fungus widely used as a model organism, the vesicle-dependent secretory mechanisms that lead to polarized growth are well studied. In contrast, biosynthesis of EVs in this fungus has been practically unexplored. In the present work, we analyzed N. crassa culture's supernatant for the presence of EVs by dynamic light scattering (DLS), transmission electron microscopy (TEM) and proteomic analysis. We identified spherical membranous structures, with a predominant subpopulation averaging a hydrodynamic diameter (dh) of 68 nm and a particle diameter (dp) of 38 nm. EV samples stained with osmium tetroxide vapors were better resolved than those stained with uranyl acetate. Mass spectrometry analysis identified 252 proteins, including enzymes involved in carbohydrate metabolic processes, oxidative stress response, cell wall organization/remodeling, and circadian clock-regulated proteins. Some of these proteins have been previously reported in exosomes from human cells or in EVs of other fungi. In view of the results, it is suggested a putative role for EVs in cell wall biosynthesis and vegetative development in N. crassa.

Beginnings of a plant parasite: early development of Rafflesia consueloae inside its Tetrastigma host.

MAIN CONCLUSION: Extensive histology of host organs revealed the early events in the vegetative growth of Rafflesia consueloae including initial infection site, endophyte distribution, and other developmental events prior to bud emergence. The early events in the vegetative development of the holoparasite Rafflesia have long remained a mystery. Because its entire vegetative growth occurs within the host body, very little is known about the developmental events prior to emergence of the floral shoot. The goal of this study was to describe the events that occur during the vegetative growth of R. consueloae, particularly in the early stages of infection. We performed extensive microtome sectioning of multiple root and stem segments from different Tetrastigma host individuals to examine the cytology, distribution, and development of the R. consueloae endophyte within the host tissues. We found that R. consueloae infection is restricted to the roots of its host. Infection begins within the vascular cambium where the endophyte appears to initially reside prior to their radial spread to the vascular tissues. The tissues obtained from different host individuals had varying degrees of infection alluding to a possible role of host resistance mechanisms and/or varying levels of parasite infectiousness. Endophyte presence in host vines without external manifestations of infection indicates that the parasite may dwell within the host tissues for prolonged periods as small cell clusters without transitioning to the reproductive stage. Furthermore, we found that floral shoots may develop in scarcely infected host tissues indicating that extensive endophyte growth within the host is not a prerequisite to the onset of reproductive development. Overall, our study describes for the first time the developmental events prior to emergence of R. consueloae buds from its host.

Poplar carbohydrate-active enzymes: whole-genome annotation and functional analyses based on RNA expression data.

Carbohydrate-active enzymes (CAZymes) catalyze the formation and modification of glycoproteins, glycolipids, starch, secondary metabolites and cell wall biopolymers. They are key enzymes for the biosynthesis of food and renewable biomass. Woody biomass is particularly important for long-term carbon storage and as an abundant renewable natural resource for many industrial applications. This study presents a re-annotation of CAZyme genes in the current Populus trichocarpa genome assembly and in silico functional characterization, based on high-resolution RNA-Seq data sets. Altogether, 1914 CAZyme and expansin genes were annotated in 101 families. About 1797 of these genes were found expressed in at least one Populus organ. We identified genes involved in the biosynthesis of different cell wall polymers and their paralogs. Whereas similar families exist in poplar and Arabidopsis thaliana (with the exception of CBM13 found only in poplar), a few families had significantly different copy numbers between the two species. To identify the transcriptional coordination and functional relatedness within the CAZymes and other proteins, we performed co-expression network analysis of CAZymes in wood-forming tissues using the AspWood database (http://aspwood.popgenie.org/aspwood-v3.0/) for Populus tremula. This provided an overview of the transcriptional changes in CAZymes during the transition from primary to secondary wall formation, and the clustering of transcripts into potential regulons. Candidate enzymes involved in the biosynthesis of polysaccharides were identified along with many tissue-specific uncharacterized genes and transcription factors. These collections offer a rich source of targets for the modification of secondary cell wall biosynthesis and other developmental processes in woody plants.

Strong temporal dynamics of QTL action on plant growth progression revealed through high-throughput phenotyping in canola.

A major challenge of plant biology is to unravel the genetic basis of complex traits. We took advantage of recent technical advances in high-throughput phenotyping in conjunction with genome-wide association studies to elucidate genotype-phenotype relationships at high temporal resolution. A diverse Brassica napus population from a commercial breeding programme was analysed by automated non-invasive phenotyping. Time-resolved data for early growth-related traits, including estimated biovolume, projected leaf area, early plant height and colour uniformity, were established and complemented by fresh and dry weight biomass. Genome-wide SNP array data provided the framework for genome-wide association analyses. Using time point data and relative growth rates, multiple robust main effect marker-trait associations for biomass and related traits were detected. Candidate genes involved in meristem development, cell wall modification and transcriptional regulation were detected. Our results demonstrate that early plant growth is a highly complex trait governed by several medium and many small effect loci, most of which act only during short phases. These observations highlight the importance of taking the temporal patterns of QTL/allele actions into account and emphasize the need for detailed time-resolved analyses to effectively unravel the complex and stage-specific contributions of genes affecting growth processes that operate at different developmental phases.

V-Mango: a functional-structural model of mango tree growth, development and fruit production.

BACKGROUND AND AIMS: Mango (Mangifera indica L.) is the fifth most widely produced fruit in the world. Its cultivation, mainly in tropical and sub-tropical regions, raises a number of issues such as the irregular fruit production across years, phenological asynchronisms that lead to long periods of pest and disease susceptibility, and the heterogeneity of fruit quality and maturity at harvest. To address these issues, we developed an integrative functional-structural plant model that synthesizes knowledge about the vegetative and reproductive development of the mango tree and opens up the possible simulation of cultivation practices. METHODS: We designed a model of architectural development in order to precisely characterize the intricate developmental processes of the mango tree. The appearance of botanical entities was decomposed into elementary stochastic events describing occurrence, intensity and timing of development. These events were determined by structural (position and fate of botanical entities) and temporal (appearance dates) factors. Daily growth and development of growth units and inflorescences were modelled using empirical distributions and thermal time. Fruit growth was determined using an ecophysiological model that simulated carbon- and water-related processes at the fruiting branch scale. KEY RESULTS: The model simulates the dynamics of the population of growth units, inflorescences and fruits at the tree scale during a growing cycle. Modelling the effects of structural and temporal factors makes it possible to simulate satisfactorily the complex interplays between vegetative and reproductive development. The model allowed the characterization of the susceptibility of mango tree to pests and the investigatation of the influence of tree architecture on fruit growth. CONCLUSIONS: This integrative functional-structural model simulates mango tree vegetative and reproductive development over successive growing cycles, allowing a precise characterization of tree phenology and fruit growth and production. The next step is to integrate the effects of cultivation practices, such as pruning, into the model.

Regulatory Mechanism of ABA and ABI3 on Vegetative Development in the Moss Physcomitrella patens.

The moss Physcomitrella patens is a model system for studying plant developmental processes. ABSCISIC ACID INSENSITIVE3 (ABI3), a transcription factor of the ABA signaling pathway, plays an important role in plant growth and development in vascular plant. To understand the regulatory mechanism of ABA and PpABI3 on vegetative development in Physcomitrella patens, we applied physiological, cellular, and RNA-seq analyses in wild type (WT) plants and ∆abi3 mutants. During ABA treatment, the growth of gametophytes was inhibited to a lesser extent ∆abi3 plants compared with WT plants. Microscopic observation indicated that the differentiation of caulonemata from chloronemata was accelerated in ∆abi3 plants when compared with WT plants, with or without 10 µM of ABA treatment. Under normal conditions, auxin concentration in ∆abi3 plants was markedly higher than that in WT plants. The auxin induced later differentiation of caulonemata from chloronemata, and the phenotype of ∆abi3 plants was similar to that of WT plants treated with exogenous indole-3-acetic acid (IAA). RNA-seq analysis showed that the PpABI3-regulated genes overlapped with genes regulated by the ABA treatment, and about 78% of auxin-related genes regulated by the ABA treatment overlapped with those regulated by PpABI3. These results suggested that ABA affected vegetative development partly through PpABI3 regulation in P. patens; PpABI3 is a negative regulator of vegetative development in P. patens, and the vegetative development regulation by ABA and PpABI3 might occur by regulating the expression of auxin-related genes. PpABI3 might be associated with cross-talk between ABA and auxin in P. patens.

Floral anatomy and vegetative development in Ceratophyllum demersum: a morphological picture of an "unsolved" plant.

PREMISE OF THE STUDY: The phylogenetic position of Ceratophyllum is still controversial in recent molecular analyses of angiosperms, with various suggestions of a sister group relation to all other angiosperms, eudicots, monocots, eudicots + monocots, and magnoliids. Therefore, the morphological characters of Ceratophyllum are important for resolving the phylogeny of angiosperms. In this study, we observed the detailed developmental anatomy of all lateral organs and their configurations to elucidate the floral development and phyllotactic pattern of Ceratophyllum demersum. METHODS: We observed fixed shoots of C. demersum with scanning electron microscopy and serial sections of the samples with light microscopy. KEY RESULTS: Bract primordia arise first, followed by the stamen primordia in staminate flowers. Both bracts and stamens initiate unidirectionally, first on the abaxial side of the floral apex and later on the adaxial side, most likely due to the contact pressure imposed by the leaf primordium at the superior node. In pistillate flowers, bract primordia on the abaxial side were also initiated first. The configuration of buds at one node showed six patterns and each pattern included at least one vegetative bud, and flower buds were always accompanied by vegetative buds at the same node. CONCLUSIONS: The initiation pattern of organs in the outer whorls of C. demersum flowers is distorted by mechanical pressure, resulting in the phyllotactic variation of staminate flowers. Vegetative buds are the main axillary buds with floral buds as accessory buds, which suggests that the shoot of C. demersum has been modified from a decussate phyllotaxis.

The FOUR LIPS and MYB88 transcription factor genes are widely expressed in Arabidopsis thaliana during development.

PREMISE OF THE STUDY: The FOUR LIPS (FLP) and MYB88 transcription factors, which are closely related in structure and function, control the development of stomata, as well as entry into megasporogenesis in Arabidopsis thaliana. However, other locations where these transcription factors are expressed are poorly described. Documenting additional locations where these genes are expressed might define new functions for these genes. METHODS: Expression patterns were examined throughout vegetative and reproductive development. The expression from two transcriptional-reporter fusions were visualized with either ß-glucuronidase (GUS) or green fluorescence protein (GFP). KEY RESULTS: Both flp and myb88 genes were expressed in many, previously unreported locations, consistent with the possibility of additional functions for FLP and MYB88. Moreover, expression domains especially of FLP display sharp cutoffs or boundaries. CONCLUSIONS: In addition to stomatal and reproductive development, FLP and MYB88, which are R2R3 MYB transcription factor genes, are expressed in many locations in cells, tissues, and organs.

Time-to-growth: photoperiod and photosynthesis make the call.

The intricate regulation of flowering time in response to day length has been extensively shown. A recent study has now revealed a similar mechanism for regulating vegetative growth. Wang et al. observed that plants measure daylength as the duration of photosynthesis and metabolite production to modulate vegetative growth.

Deciphering structural and temporal interplays during the architectural development of mango trees.

Plant architecture is commonly defined by the adjacency of organs within the structure and their properties. Few studies consider the effect of endogenous temporal factors, namely phenological factors, on the establishment of plant architecture. This study hypothesized that, in addition to the effect of environmental factors, the observed plant architecture results from both endogenous structural and temporal components, and their interplays. Mango tree, which is characterized by strong phenological asynchronisms within and between trees and by repeated vegetative and reproductive flushes during a growing cycle, was chosen as a plant model. During two consecutive growing cycles, this study described vegetative and reproductive development of 20 trees submitted to the same environmental conditions. Four mango cultivars were considered to assess possible cultivar-specific patterns. Integrative vegetative and reproductive development models incorporating generalized linear models as components were built. These models described the occurrence, intensity, and timing of vegetative and reproductive development at the growth unit scale. This study showed significant interplays between structural and temporal components of plant architectural development at two temporal scales. Within a growing cycle, earliness of bud burst was highly and positively related to earliness of vegetative development and flowering. Between growing cycles, flowering growth units delayed vegetative development compared to growth units that did not flower. These interplays explained how vegetative and reproductive phenological asynchronisms within and between trees were generated and maintained. It is suggested that causation networks involving structural and temporal components may give rise to contrasted tree architectures.

An optimized pipeline for live imaging whole Arabidopsis leaves at cellular resolution.

BACKGROUND: Live imaging is the gold standard for determining how cells give rise to organs. However, tracking many cells across whole organs over large developmental time windows is extremely challenging. In this work, we provide a comparably simple method for confocal live imaging entire Arabidopsis thaliana first leaves across early development. Our imaging method works for both wild-type leaves and the complex curved leaves of the jaw-1D mutant. RESULTS: We find that dissecting the cotyledons, affixing a coverslip above the samples and mounting samples with perfluorodecalin yields optimal imaging series for robust cellular and organ level analysis. We provide details of our complementary image processing steps in MorphoGraphX software for segmenting, tracking lineages, and measuring a suite of cellular properties. We also provide MorphoGraphX image processing scripts we developed to automate analysis of segmented images and data presentation. CONCLUSIONS: Our imaging techniques and processing steps combine into a robust imaging pipeline. With this pipeline we are able to examine important nuances in the cellular growth and differentiation of jaw-D versus WT leaves that have not been demonstrated before. Our pipeline is approachable and easy to use for leaf development live imaging.

Function of the Mitochondrial Transport Protein BcMtp1 in Regulating Vegetative Development, Asexual Reproduction, Stress Response, Fungicide Sensitivity, and Virulence of Botrytis cinerea.

In model fungi, mitochondrial transport proteins (MTPs), also known as "mitochondrial carriers" (MC), are known to facilitate the exchange of biochemical substances across the mitochondrial inner membrane. In this study, we characterized an MTP in Botrytis cinerea homologous to the known MTPs in Saccharomyces cerevisiae designated BcMtp1. The BcMtp1 deletion mutant phenotype was strikingly defective in vegetative development, conidiation, and sclerotia production. In addition, ΔBcMtp1 showed increased sensitivity to osmotic stress, oxidative stress, and cell wall biogenesis inhibitors. In the pathogenicity assay, ΔBcMtp1 displayed compromised virulence on various host-plant tissues. The BcMtp1 deletion mutant phenotype was rescued by transforming the wild-type BcMtp1 variant into the mutant. Together, these data indicate that BcMtp1 is critical for vegetative development, asexual reproduction, stress tolerance, and virulence of B. cinerea.

Identification of trehalose-6-phosphate synthase (TPS)-coding genes involved in flowering induction of Lilium× formolongi.

Lilium × formolongi, a facultative long-day (LD) plant, can complete the floral transition within one year after sowing under LD conditions. In addition to the photoperiod, the molecular mechanisms by which other flowering regulators, such as sugar, participate in juvenile development and flowering induction in L. × formolongi remain elusive. Therefore, based on the investigation of seedling development under different day length conditions, we explored the growth and nonstructural carbohydrate (NSC) contents of leaves and underground bulbs. Furthermore, the expression profiles of trehalose-6-phosphate synthase (TPS)-coding genes, LfTPSs, and miR156 were also determined. Three putative LfTPS genes, LfTPS1, LfTPS3 and LfTPS5, displayed high expression levels at the juvenile vegetative stage under different day length conditions. Among them, LfTPS1 maintained gradually elevated expression until the visible bud stage under short-day (SD) conditions. Additionally, the expression levels of LfTPS3 and LfTPS5 increased with the exogenous sucrose concentration. In contrast, the expression of miR156 rapidly decreased under the same sucrose treatments. Overexpression of LfTPS1/3/5 hastened flowering and the decline in miR156 expression levels to varying degrees in transgenic Arabidopsis. Taken together, the results demonstrate that LfTPS1, LfTPS3 and LfTPS5 modulate both juvenile vegetative development and flowering induction controlled by sugars in L. × formolongi.

Plant GDSL Esterases/Lipases: Evolutionary, Physiological and Molecular Functions in Plant Development.

GDSL esterases/lipases (GELPs), present throughout all living organisms, have been a very attractive research subject in plant science due mainly to constantly emerging properties and functions in plant growth and development under both normal and stressful conditions. This review summarizes the advances in research on plant GELPs in several model plants and crops, including Arabidopsis, rice, maize and tomato, while focusing on the roles of GELPs in regulating plant development and plant-environment interactions. In addition, the possible regulatory network and mechanisms of GELPs have been discussed.

Characterizing the gene-environment interaction underlying natural morphological variation in Neurospora crassa conidiophores using high-throughput phenomics and transcriptomics.

Neurospora crassa propagates through dissemination of conidia, which develop through specialized structures called conidiophores. Recent work has identified striking variation in conidiophore morphology, using a wild population collection from Louisiana, United States of America to classify 3 distinct phenotypes: Wild-Type, Wrap, and Bulky. Little is known about the impact of these phenotypes on sporulation or germination later in the N. crassa life cycle, or about the genetic variation that underlies them. In this study, we show that conidiophore morphology likely affects colonization capacity of wild N. crassa isolates through both sporulation distance and germination on different carbon sources. We generated and crossed homokaryotic strains belonging to each phenotypic group to more robustly fit a model for and estimate heritability of the complex trait, conidiophore architecture. Our fitted model suggests at least 3 genes and 2 epistatic interactions contribute to conidiophore phenotype, which has an estimated heritability of 0.47. To uncover genes contributing to these phenotypes, we performed RNA-sequencing on mycelia and conidiophores of strains representing each of the 3 phenotypes. Our results show that the Bulky strain had a distinct transcriptional profile from that of Wild-Type and Wrap, exhibiting differential expression patterns in clock-controlled genes (ccgs), the conidiation-specific gene con-6, and genes implicated in metabolism and communication. Combined, these results present novel ecological impacts of and differential gene expression underlying natural conidiophore morphological variation, a complex trait that has not yet been thoroughly explored.

Diversity and Agronomic Performance of Lupinus mutabilis Germplasm in European and Andean Environments.

The introduction of Lupinus mutabilis (Andean lupin) in Europe will provide a new source of protein and oil for plant-based diets and biomass for bio-based products, while contributing to the improvement of marginal soils. This study evaluates for the first time the phenotypic variability of a large panel of L. mutabilis accessions both in their native environment and over two cropping conditions in Europe (winter crop in the Mediterranean region and summer crop in North-Central Europe), paving the way for the selection of accessions adapted to specific environments. The panel of 225 accessions included both germplasm pools from the Andean region and breeding lines from Europe. Notably, we reported higher grain yield in Mediterranean winter-cropping conditions (18 g/plant) than in the native region (9 g/plant). Instead, North European summer-cropping conditions appear more suitable for biomass production (up to 2 kg/plant). The phenotypic evaluation of 16 agronomical traits revealed significant variation in the panel. Principal component analyses pointed out flowering time, yield, and architecture-related traits as the main factors explaining variation between accessions. The Peruvian material stands out among the top-yielding accessions in Europe, characterized by early lines with high grain yield (e.g., LIB065, LIB072, and LIB155). Bolivian and Ecuadorian materials appear more valuable for the selection of genotypes for Andean conditions and for biomass production in Europe. We also observed that flowering time in the different environments is influenced by temperature accumulation. Within the panel, it is possible to identify both early and late genotypes, characterized by different thermal thresholds (600°C-700°C and 1,000-1,200°C GDD, respectively). Indications on top-yielding and early/late accessions, heritability of morpho-physiological traits, and their associations with grain yield are reported and remain largely environmental specific, underlining the importance of selecting useful genetic resources for specific environments. Altogether, these results suggest that the studied panel holds the genetic potential for the adaptation of L. mutabilis to Europe and provide the basis for initiating a breeding program based on exploiting the variation described herein.

DEFECTIVE EMBRYO AND MERISTEMS1 (DEM1) Is Essential for Cell Proliferation and Cell Differentiation in Tomato.

Most flowering plant species contain at least two copies of the DEFECTIVE EMBRYO AND MERISTEMS (DEM) gene with the encoded DEM proteins lacking homology to proteins of known biochemical function. In tomato (Sl; Solanum lycopersicum), stable mutations in the SlDEM1 locus result in shoot and root meristem defects with the dem1 mutant failing to progress past the cotyledon stage of seedling development. Generation of a Somatic Mutagenesis of DEM1 (SMD) transformant line in tomato allowed for the characterization of SlDEM1 gene function past the seedling stage of vegetative development with SMD plants displaying a range of leaf development abnormalities. Further, the sectored or stable in planta expression of specific regions of the SlDEM1 coding sequence also resulted in the generation of tomato transformants that displayed a range of vegetative development defects, which when considered together with the dem1 mutant seedling and SMD transformant line phenotypic data, allowed for the assignment of SlDEM1 gene function to early embryo development, adaxial epidermis cell development, lateral leaf blade expansion, and mesophyll cell proliferation and differentiation.

Phenylpropanoid Biosynthesis Gene Expression Precedes Lignin Accumulation During Shoot Development in Lowland and Upland Switchgrass Genotypes.

Efficient conversion of lignocellulosic biomass into biofuels is influenced by biomass composition and structure. Lignin and other cell wall phenylpropanoids, such as para-coumaric acid (pCA) and ferulic acid (FA), reduce cell wall sugar accessibility and hamper biochemical fuel production. Toward identifying the timing and key parameters of cell wall recalcitrance across different switchgrass genotypes, this study measured cell wall composition and lignin biosynthesis gene expression in three switchgrass genotypes, A4 and AP13, representing the lowland ecotype, and VS16, representing the upland ecotype, at three developmental stages [Vegetative 3 (V3), Elongation 4 (E4), and Reproductive 3 (R3)] and three segments (S1-S3) of the E4 stage under greenhouse conditions. A decrease in cell wall digestibility and an increase in phenylpropanoids occur across development. Compared with AP13 and A4, VS16 has significantly less lignin and greater cell wall digestibility at the V3 and E4 stages; however, differences among genotypes diminish by the R3 stage. Gini correlation analysis across all genotypes revealed that lignin and pCA, but also pectin monosaccharide components, show the greatest negative correlations with digestibility. Lignin and pCA accumulation is delayed compared with expression of phenylpropanoid biosynthesis genes, while FA accumulation coincides with expression of these genes. The different cell wall component accumulation profiles and gene expression correlations may have implications for system biology approaches to identify additional gene products with cell wall component synthesis and regulation functions.

Heterologous Expression of the Transcription Factor EsNAC1 in Arabidopsis Enhances Abiotic Stress Resistance and Retards Growth by Regulating the Expression of Different Target Genes.

Heterologous expression of a transcription factor (TF) gene in a related species is a useful method for crop breeding and the identification of gene function. The differences in phenotype and target gene expression between HE lines (with the heterologous expression of an ortholog) and OX lines (with an overexpressed native gene) must be understood. EsNAC1, encoding a NAC protein and the ortholog of RD26 in Arabidopsis, was cloned from Eutrema salsugineum and introduced into Arabidopsis. The heterologous expression of EsNAC1 retarded the vegetative growth of Arabidopsis, and the transgenic plants (HE lines) showed much greater resistance to salt and oxidative stress than the wild type, Col-0. The HE lines accumulated 2.8-fold (8-h light) of starch, 1.42-fold of Chlorophyll a and 1.31-fold of Chlorophyll b than Col-0 during the light period, with obvious differences compared to the RD26OX line. A genome-wide ChIP (chromatin immunoprecipitation analysis)-on-chip assay revealed that EsNAC1 targeted promoters of different genes compared to RD26. In HE lines, EsNAC1 could specifically upregulate the expression level of TF genes NAC DOMAIN CONTAINING PROTEIN 62 (ANAC062), INTEGRASE-TYPE DNA-BINDING PROTEIN (TINY2), and MYB HYPOCOTYL ELONGATION-RELATED (MYBH) to show more effective abiotic stress resistance than RD26OX lines. Moreover, DELTA1-PYRROLINE-5-CARBOXYLATE SYNTHASE 1 (P5CS1), TRYPTOPHAN BIOSYNTHESIS 2 (TRP2) or GALACTINOL SYNTHASE 2 (GOLS2), was also specifically regulated by EsNAC1 to retard the vegetative growth of HE lines, but not the brassinosteroid singling pathway in RD26OX lines. These differences in phenotypes and metabolism between the HE lines and the RD26OX line implied that the differential features could be produced from the diversity of target genes in the transgenic plants when the ortholog was introduced.

Kinesin-4 Functions in Vesicular Transport on Cortical Microtubules and Regulates Cell Wall Mechanics during Cell Elongation in Plants.

In plants, anisotropic cell expansion depends on cortical microtubules that serve as tracks along which macromolecules and vesicles are transported by the motor kinesins of unknown identities. We used cotton (Gossypium hirsutum) fibers that underwent robust elongation to discover kinesins that are involved in cell elongation and found Gh KINESIN-4A expressed abundantly. The motor was detected by immunofluorescence on vesicle-like structures that were associated with cortical microtubules. In Arabidopsis thaliana, the orthologous motor At KINESIN-4A/FRA1, previously implicated in cellulose deposition during secondary growth in fiber cells, was examined by live-cell imaging in cells expressing the fluorescently tagged functional protein. The motor decorated vesicle-like particles that exhibit a linear movement along cortical microtubules with an average velocity of 0.89 µm/min, which was significantly different from those linked to cellulose biosynthesis. We also discovered that At KINESIN-4A/FRA1 and the related At KINESIN-4C play redundant roles in cell wall mechanics, cell elongation, and the axial growth of various vegetative and reproductive organs, as the loss of At KINESIN-4C greatly enhanced the defects caused by a null mutation at the KINESIN-4A/FRA1 locus. The double mutant displayed a lack of cell wall softening at normal stages of rapid cell elongation. Furthermore, enhanced deposition of arabinose-containing carbohydrate was detected in the kinesin-4 mutants. Our findings established a connection between the Kinesin-4-based transport of cargoes containing non-cellulosic components along cortical microtubules and cell wall mechanics and cell elongation in flowering plants.