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bioRxiv2018

MyROOT: A novel method and software for the semi-automatic measurement of plant root length

Root analysis is essential for both academic and agricultural research. Despite the great advances in root phenotyping and imaging however, calculating root length is still performed manually and involves considerable amounts of labor and time. To overcome these limitations, we have developed MyROOT, a novel software for the semi-automatic quantification of root growth of seedlings growing directly in agar plates. Our method automatically determines the scale from the image of the plate, and subsequently measures the root length of the individual plants. To this aim, MyROOT combines a bottom-up root tracking approach with a hypocotyl detection algorithm. At the same time as providing accurate root measurements, MyROOT also significantly minimizes the user intervention required during the process. Using Arabidopsis, we tested MyROOT with seedlings from different growth stages. Upon comparing the data obtained using this software with that of manual root measurements, we found that there are no significant differences (t-test, p-value < 0.05). Thus, MyROOT will be of great aid to the plant science community by permitting high-throughput root length measurements while saving on both labor and time.

plant biology↗

bioRxiv2018

Temporal Dynamics of the Origin and Domestication of Sweet Potato and Implications for Dispersal to Polynesia

In a study focussed on the origin of Ipomoea batatas (L.) Lam. (the sweet potato) and its close relatives [1], the authors claimed that a specimen of sweet potato collected by Banks and Solander in 1769 in Polynesia was distinct and along with some other varieties had levels of genetic variation of more than 100,000 years. This timeframe, along with several other lines of evidence, was interpreted, by the authors, as questioning human-mediated transport of the sweet potato to Polynesia within the last 1,000 years. This aspect of the paper received widespread media coverage as well as a number of critical remarks, and correspondence. The criticism questioned whether claims in the paper were justified. Here, three authors from the paper take the opportunity to fully explain the reasoning for calling into question the need to invoke human mediated transport of the sweet potato to Polynesia.

plant biology↗

bioRxiv2018

PII1: a protein involved in starch initiation that determines granule number and size in Arabidopsis chloroplast.

The initiation of starch granule formation is still poorly understood. However, soluble starch synthase 4 (SS4) appears to be a major component of this process since it is required to synthetize the correct number of starch granules in the chloroplasts of Arabidopsis thaliana plants. A yeast-2-hybrid screen allowed the identification of several putative SS4 interacting partners. We identified the product of At4g32190 locus as a chloroplast-targeted PROTEIN INVOLVED IN STARCH INITIATION (named PII1). Arabidopsis mutants devoid of PII1 display an alteration of starch initiation process and accumulate, on average, one starch granule per plastid instead of the 5 to 7 granules found in plastids of wild-type plants. These granules are larger than in wild type and they remain flat and lenticular. pii1 mutants display wild-type growth rates and accumulate standard starch amounts. Moreover, starch characteristics, such as amylopectin chain length distribution, remain unchanged. Our results reveal the involvement of PII1 in starch priming process in Arabidopsis leaves through interaction with SS4.

plant biology↗

bioRxiv2018

Investigating the effect of Target of Rapamycin kinase inhibition on the Chlamydomonas reinhardtii phosphoproteome: from known homologs to new targets

O_LITarget of Rapamycin (TOR) kinase is a conserved regulator of cell growth whose activity is modulated in response to nutrients, energy and stress. Key proteins involved in the pathway are conserved in the model photosynthetic microalga Chlamydomonas reinhardtii, but the substrates of TOR kinase and downstream signaling network have not been elucidated. Our study provides a new resource for investigating the phosphorylation networks governed by the TOR kinase pathway in Chlamydomonas.\nC_LIO_LIWe used quantitative phosphoproteomics to investigate the effects of inhibiting Chlamydomonas TOR kinase on dynamic protein phosphorylation. Wild-type and AZD-insensitive Chlamydomonas strains were treated with TOR-specific chemical inhibitors (rapamycin, AZD8055 and Torin1), after which differentially affected phosphosites were identified.\nC_LIO_LIOur quantitative phosphoproteomic dataset comprised 2,547 unique phosphosites from 1,432 different proteins. Inhibition of TOR kinase caused significant quantitative changes in phosphorylation at 258 phosphosites, from 219 unique phosphopeptides.\nC_LIO_LIOur results include Chlamydomonas homologs of TOR signaling-related proteins, including a site on RPS6 with a decrease in phosphorylation. Additionally, phosphosites on proteins involved in translation and carotenoid biosynthesis were identified. Follow-up experiments guided by these phosphoproteomic findings in lycopene beta/epsilon cyclase showed that carotenoid levels are affected by TORC1 inhibition and carotenoid production is under TOR control in algae.\nC_LI

plant biology↗

bioRxiv2018

Biogenesis of a young, 22-nt microRNA in Phaseoleae species by precursor-programmed uridylation

Phased, secondary siRNAs (phasiRNAs) represent a class of small RNAs in plants generated via distinct biogenesis pathways, predominantly dependent on the activity of 22 nt miRNAs. Most 22 nt miRNAs are processed by DCL1 from miRNA precursors containing an asymmetric bulge, yielding a 22/21 nt miRNA/miRNA* duplex. Here we show that miR1510, a soybean miRNA capable of triggering phasiRNA production from numerous NB-LRRs, previously described as 21 nt in its mature form, primarily accumulates as a 22 nt isoform via monouridylation. We demonstrate that in Arabidopsis, this uridylation is performed by HESO1. Biochemical experiments showed that the 3 terminus of miR1510 is only partially 2-O-methylated, because of the terminal mispairing in the miR1510/miR1510* duplex that inhibits HEN1 activity in soybean. miR1510 emerged in the Phaseoleae ~41 to 42 MYA with a conserved precursor structure yielding a 22 nt monouridylated form, yet a variant in mung bean is processed directly in a 22 nt mature form. This analysis of miR1510 yields two observations: (1) plants can utilize post-processing modification to generate abundant 22 nt miRNA isoforms to more efficiently regulate target mRNA abundances; (2) comparative analysis demonstrates an example of selective optimization of precursor processing of a young plant miRNA.

plant biology↗

bioRxiv2018

N-1-naphthylphthalamic acid stimulates tomato hypocotyl elongation, elevates ethylene levels and alters metabolic homeostasis

One sentence summaryN-1-naphthylphthalamic acid (NPA) treatment stimulates tomato hypocotyl elongation likely by elevating ethylene emission and lowering indole-3-butyric acid levels in the seedlings.\n\nAbstractIn higher plants, phytohormone indole-3-acetic acid is characteristically transported from the apex towards the base of the plant, termed as polar auxin transport (PAT). Among the inhibitors blocking PAT, N-1-naphthylphthalamic acid (NPA) that targets ABCB transporters is most commonly used. NPA-treated light-grown Arabidopsis seedlings show severe inhibition of hypocotyl and root elongation. In light-grown tomato seedlings, NPA inhibited root growth, but contrary to Arabidopsis stimulated hypocotyl elongation. The NPA-stimulation of hypocotyl elongation was milder in blue, red, and far-red light-grown seedlings. The NPA-treatment stimulated emission of ethylene from the seedlings. The scrubbing of ethylene by mercuric perchlorate reduced NPA-stimulated hypocotyl elongation. NPA action on hypocotyl elongation was antagonized by 1-methylcyclopropene, an inhibitor of ethylene action. NPA-treated seedlings had reduced levels of indole-3-butyric acid and higher levels of zeatin in the shoots. NPA did not alter indole-3-acetic levels in shoots. The analysis of metabolic networks indicated that NPA-treatment induced moderate shifts in the networks compared to exogenous ethylene that induced a drastic shift in metabolic networks. Our results indicate that in addition to ethylene, NPA-stimulated hypocotyl elongation in tomato may also involve zeatin and indole-3-butyric acid. Our results indicate that NPA-mediated physiological responses may vary in a species-specific fashion.

plant biology↗

bioRxiv2018

Connecting the dots between computational tools to analyse soil-root water relations

In the recent years, many computational tools, such as image analysis, data management, process-based simulation and upscaling tools, were developed to help quantify and understand water flow in the soil-root system, at multiple scales (tissue, organ, plant and population). Several of these tools work together or, at least, are compatible. However, for the un-informed researcher, they might seem disconnected, forming a unclear and disorganised succession of tools.\n\nIn this article, we present how different pieces of work can be further developed by connecting them to analyse soil-root-water relations in a comprehensive and structured network. This \"explicit network of soil-root computational tools\" informs the reader about existing tools and help them understand how their data (past and future) might fit within the network. We also demonstrate the novel possibilities of scale-consistent parameterizations made possible by the network with a set of case studies from the literature. Finally, we discuss existing gaps in the network and how we can move forward to fill them.\n\nHighlightsMany computational tools exist to quantify water flow in the soil-root system. These tools can be arranged in a comprehensive network that can be leveraged to better interpret experimental data.

plant biology↗

bioRxiv2018

Modulation of TOR kinase activity in Chlamydomonas reinhardtii: Effect of N-starvation and changing carbon pool

Target Of Rapamycin (TOR) kinase is a sensor as well as a central integrator of internal and external metabolic cues. The upstream and downstream signals of this kinase are very well characterized in animals. However, in algae, higher plants and other photosynthetic organisms, the components of the TOR kinase-signaling are yet to be characterized. Here, we establish an assay system to study TOR kinase activity in C.reinhardtii using the phosphorylation status of its downstream target, CrS6K. We further use this assay to monitor TOR kinase activity under various physiological states such as photoautotrophy, heterotrophy, mixotrophy and nitrogen starvation. We observe that autotrophy in light (and not in dark) leads to TOR kinase attenuation during N starvation while the same is not observed in mixotrophy. Importantly, we show that the external carbon source glucose is sensed and uptaken by C.reinhardtii cells only in the presence of light and not in the dark. And such exogenously added glucose, as the photoassimilate carbon mimic, results in enhanced production of ROS, induction of autophagy and concomitant drop in TOR kinase activity, creating N-starvation-like cellular state even in N+ conditions. Interestingly, dose dependent addition of glucose revealed TOR kinase activation in low glucose regime (ROS independent) followed by attenuation of TOR kinase (ROS dependent) at high glucose levels.\n\nSummaryTOR kinase activity in C.reinhardtii is modulated by available carbon source especially glucose, where low levels of glucose cause an increase whereas high levels cause a reduction in TOR kinase activity.

plant biology↗

bioRxiv2018

Autophagy is essential for optimal Fe translocation to seeds in Arabidopsis

Micronutrient deficiencies affect a large part of the world population. They are mostly due to the consumption of grains with insufficient content of Fe or Zn. Both de novo uptake by roots and recycling from leaves may provide seeds with nutrients. Autophagy, which is a conserved mechanism for nutrient recycling in eukaryotes, was shown to be involved in nitrogen remobilization to seeds. Here, we have investigated the role of this mechanism in micronutrient translocation to seeds. We found that several Arabidopsis thaliana plants impaired in autophagy display defects in nutrient remobilization to seeds. In atg5-1 mutant, which is completely defective in autophagy, the efficiency of Fe translocation from vegetative organs to seeds was severely decreased even when Fe was provided during seed formation. Combining atg5-1 with sid2 mutation that counteracts premature senescence associated to autophagy deficiency and using 57Fe pulse labeling, we could propose a two-step mechanism in which iron taken up de novo during seed formation is first accumulated in vegetative organs and subsequently remobilized to seeds. Finally, we showed that translocations of zinc and manganese to seeds are also dependent on autophagy. Fine tuning autophagy during seed formation opens therefore new possibilities to improve micronutrient remobilization to seeds.

plant biology↗

bioRxiv2018

Intraspecific Polymorphism for the Presence and Absence of Entire Mitochondrial Chromosomes

Although mitochondrial genomes are typically thought of as single circular molecules, these genomes are fragmented into multiple chromosomes in many eukaryotes, raising intriguing questions about inheritance and (in)stability of mtDNA in such systems. A previous comparison of mitochondrial genomes from two different individuals of the angiosperm species Silene noctiflora found variation in the presence of entire mitochondrial chromosomes. Here, we expand on this work with a geographically diverse sampling of 25 S. noctiflora populations. We also included the closely related species S. turkestanica and S. undulata, with the latter exhibiting a surprising phylogenetic placement nested within the diversity of S. noctiflora mitochondrial haplotypes. Using a combination of deep sequencing and PCR-based screening for the presence of 22 different mitochondrial chromosomes, we found extensive variation in the complement of chromosomes across individuals. Much of this variation could be attributed to recent chromosome loss events. Despite the fragmented structures of these mitochondrial genomes and the evidence for occasional biparental inheritance in other Silene species, we did not find any indication of recombination between distinct mitochondrial haplotypes either within or among mitochondrial chromosomes, which may reflect the extreme paucity of nucleotide sequence polymorphism and/or the high selfing rate in this species. These results suggest that the massively expanded and fragmented mitochondrial genomes of S. noctiflora may have entered a phase of genome reduction in which they are losing entire chromosomes at a rapid rate.

plant biology↗

bioRxiv2018

Histone deacetylase HDA19 affects cortical cell fate by interacting with SCARECROW in the Arabidopsis root

Abstract/SummaryThe Arabidopsis root epidermis is a simple model for investigating cell fate specification and pattern formation. In addition to regulatory networks consisting of transcription factors, histone deacetylases are also involved in the cellular patterning process. Here we report HDA19 affects the root epidermal cellular pattern through regulation of cortical cell fate by interacting with SCARECROW. This work reveals two new components in cortical cell specification and uncovers a new facet of SCR function.

plant biology↗

bioRxiv2018

Choline monooxygenase transcript expression triggers glycine betaine accumulation in Suaeda maritima when subjected to a salt concentration optimal for its growth

Suaeda maritima(L.) Dumort, an annual halophyte known to be a salt-accumulator is also known for the accumulation of the osmolyte, glycine betaine (GB). This study is an attempt to understand the growth and GB accumulation under optimal concentration of 200 mM NaCl in S. maritima. Salt treatment with 200 mM NaCl showed a significant increase in shoot growth after two weeks. The shoots appeared succulent and turgid after two weeks of salt treatment compared to that of the control which appeared slender and wilted. The treated seedlings also exhibited a significant increase in GB content after two weeks of salt treatment. In order to determine the molecular basis of GB accumulation, qRT PCR of three key genes involved in the pathway, choline monooxygenase, betaine aldehyde dehydrogenase and phospho-ethanolamine N-methyl transferase was performed. Transcript level expression of the three genes revealed a high up-regulation of choline monooxygeanse transcripts when compared to that of the other two transcripts at two days of salt treatment. The results indicate that S. maritima requires salt for its growth and is a natural accumulator of GB. Although all the three genes were salt inducible, the high up-regulation of choline monooxygenase greatly contributes to the accumulation of GB under optimal growth as well as NaCl concentration.\n\nAbbreviations

plant biology↗

bioRxiv2018

Real time portable genome sequencing for global food security

The United Nations has listed Zero Hunger as one of the 17 global sustainable development goals to end extreme poverty by 2030. Plant viruses are a major constraint to crop production globally causing an estimated $30 billion in damage 1 leaving millions of people food insecure 2. In Africa, agriculture employs up to 50% of the workforce, yet only contributes 15% to the GDP on average 3, suggesting that there is low productivity and limited value addition. This can be addressed through continued innovation in the fields of science and technology as suggested in the Science Agenda for Agriculture in Africa (S3A) 4. Sustainable management of plant viruses and their associated vectors must include efficient diagnostics for surveillance, detection and identification to inform disease management, including the development and strategic deployment of virus resistant varieties. To date, researchers have been utilizing conventional methods such as; PCR, qPCR, high throughput sequencing (RNA-Seq, DNA-Seq) and Sanger sequencing for pathogen identification. However, these methods are both costly and time consuming, delaying timely control actions. The emergence of new tools for real-time diagnostics, such as the Oxford Nanopore MinION, have recently proven useful for early detection of Ebola 6 and Zika 7,8, even in low resourced laboratories. For the first time globally, the MinION portable pocket DNA sequencer was used to sequence whole plant virus genomes. We used this technology to identify the begomoviruses causing the devastating CMD which is ravaging smallholder farmers crops in sub-Saharan Africa. Cassava, a carbohydrate crop from which tapioca originates, is a major source of calories for over eight hundred (800) million people worldwide. With this technology, farmers struggling with diseased crops can take immediate, restorative action to improve their livelihoods based on information about the health of their plants, generated using a portable, real-time DNA sequencing device.

plant biology↗

bioRxiv2018

CRISPR-Cas9 interference in cassava linked to the evolution of editing-resistant geminiviruses

We used CRISPR-Cas9 in the staple food crop cassava with the aim of engineering resistance to African cassava mosaic virus, a member of a widespread and important family of plant-pathogenic DNA viruses. We found that between 33 and 48% of edited virus genomes evolved a conserved single-nucleotide mutation that confers resistance to CRISPR-Cas9 cleavage. Our study highlights the potential for virus escape from this technology. Care should be taken to design CRISPR-Cas9 experiments that minimize the risk of virus escape.

plant biology↗

bioRxiv2018

Evidence of a Novel Vitivirus in Grapevine cv Riesling

Vitivirus are ssRNA(+) viruses in the family Betaflexiviridae (subfamily Trivirinae). There are currently ten ICTV recognized virus species in the genus; nevertheless, the extended use of NGS technologies is rapidly expanding their diversity and six more have been proposed recently. Here, we present the characterization of a novel virus from grapevines, which fits the genomic architecture and evolutionary constraints to be classifiable within the Vitivirus genus. The detected virus sequence is 7,607 nt long, including a typical genome organization of ORFs encoding a replicase (RP), a 22 kDa protein, a movement protein, a coat protein (CP) and a nucleic acid binding protein. Here, we present the characterization of a novel virus from grapevines. Phylogenetic analyses based on the predicted RP and CP protein unequivocally places the new virus within the Vitivirus genus. Multiple independent RNAseq data confirmed the presence of the detected virus in berries at diverse developmental stages. Additionally, we detected, confirmed, and assembled virus sequences from grapevine samples of distinct cultivars from America, Europe, Asia and Oceania, sharing 74.9%-97.9% nt identity, suggesting that the identified virus is widely distributed and diverse. We propose the name grapevine virus L (GVL) to the detected Vitivirus.

plant biology↗

bioRxiv2018

Long-day increase of HvVRN2 expression marks the deadline to fulfill the vernalization requirement in winter barley

Vernalization and photoperiod cues are integrated in winter barley plants to flower in the right conditions. We hypothesize that there is a timeframe to satisfy the vernalization needs in order to flower in the optimum moment. Growth and expression of different flowering promoters (HvVRN1, HvCO2, Ppd-H1, HvFT1, HvFT3) and repressors (HvVRN2, HvCO9 and HvOS2) were evaluated in two winter barley varieties under: (1) natural increasing photoperiod, without vernalization, and (2) under short day conditions in three insufficient vernalization treatments. Here, we provide evidence of the existence of a day-length threshold, around 12 h 30 min in our latitudes (Zaragoza, Spain, 41{degrees}43N), marked by the rise of HvVRN2 expression, which defines the moment in which cold requirement must be satisfied to acquire competency to flower. Before that, expression of HvCO2 was induced and might be promoting HvFT1 in both inductive and non-inductive conditions. HvFT3, to be effectively expressed, must receive induction of cold or plant development, through downregulation of HvVRN2 and HvOS2. We emphasize the contribution of HvOS2, together with HvVRN2, in the delay of flowering in vernalization-responsive cultivars. Understanding this complex mechanism of flowering might be useful for breeders to define varieties, particularly in a climate change scenario.

plant biology↗

bioRxiv2018

High-throughput Analysis of Arabidopsis Stem Vibrations to Identify Mutants with Altered Mechanical Properties

Mechanical properties are rarely used as quantitative indices for the large-scale mutant screening of plants, even in the model plant Arabidopsis thaliana. The mechanical properties of plant stems generally influence their vibrational characteristics. Here, we developed Python-based software, named AraVib, for the high-throughput analysis of free vibrations of plant stems, focusing specifically on Arabidopsis stem vibrations, and its extended version, named AraVibS, to identify mutants with altered mechanical properties. These programs can be used without knowledge of Python and require only an inexpensive handmade setting stand and an iPhone/iPad with a high-speed shooting function for data acquisition. Using our system, we identified an nst1 nst3 double-mutant lacking secondary cell walls in fiber cells and a wrky12 mutant displaying ectopic formation of secondary cell wall compared with wild type by employing only two growth traits (stem height and fresh weight) in addition to videos of stem vibrations. Furthermore, we calculated the logarithmic decrement, the damping ratio, the natural frequency and the stiffness based on the spring-mass-damper model from the video data using AraVib. The stiffness was estimated to be drastically decreased in nst1 nst3, which agreed with previous tensile test results. However, in wrky12, the stiffness was significantly increased. These results demonstrate the effectiveness of our new system. Because our method can be applied in a high-throughput manner, it can be used to screen for mutants with altered mechanical properties.

plant biology↗

bioRxiv2018

Anisotropic growth is achieved through the additive mechanical effect of material anisotropy and elastic asymmetry

Fast directional growth is a necessity for the young seedling: after germination, the seedling needs to quickly reach through the soil to begin its autotrophic life. In most dicot plants, this rapid escape is due to the anisotropic elongation of the hypocotyl, the columnar organ between the root and the shoot meristems. Such anisotropic growth is common in many plant organs and is canonically attributed to cell wall anisotropy produced by oriented cellulose fibers in the cell wall. More recently, a mechanism based on asymmetric cell wall elasticity has been proposed, produced by differential pectin biochemistry. Here we present a harmonizing model for anisotropic growth control in the dark-grown Arabidopsis hypocotyl: basic anisotropic information is provided by cellulose orientation (proxied by microtubules) and additive anisotropic information is provided by pectin-based elastic asymmetry in the epidermis. We demonstrate that hypocotyl growth was always anisotropic with axial and transverse walls growing differently, from germination. We present experimental evidence for pectin biochemical differences and wall mechanics underlying this differential growth. We demonstrate that pectin biochemical changes control the transition to rapid growth characteristic of Arabidopsis hypocotyl elongation, and provide evidence for a substantial mechanical role for pectin in the cell wall when microtubules are compromised. Lastly, our in silico modelling experiments indicate an additive combination for pectin biochemistry and cellulose orientation in promoting anisotropic growth.

plant biology↗