Reduced tillering and dwarfing genes alter root traits and rhizo-economics in wheat.

The tiller inhibition (tin) and Reduced height (Rht) genes strongly influence the carbon partitioning and architecture of wheat shoots, but their effects on the energy economy of roots have not been examined in detail. We examined multiple root traits in three sets of near-isogenic wheat lines (NILs) that differ in the tin gene or various dwarfing gene alleles (Rht-B1b, Rht-D1b, Rht-B1c and Rht-B1b + Rht-D1b) to determine their effects on root structure, anatomy and carbon allocation. The tin gene resulted in fewer tillers but more costly roots in an extreme tin phenotype with a Banks genetic background due to increases in root-to-shoot ratio, total root length, and whole root respiration. However, this effect depended on the genetic background as tin caused both smaller shoots and roots in a different genetic background. The semi-dwarf gene Rht-B1b caused few changes to the root structure, whereas Rht-D1b, Rht-B1c and the double dwarf (Rht-B1b + Rht-D1b) decreased the root biomass. Rht-B1c reduced the energy cost of roots by increasing specific root length, increasing the volume of cortical aerenchyma and by reducing root length, number, and biomass without affecting the root-to-shoot ratio. This work informs researchers using tin and Rht genes how to modify root system architecture to suit specific environments.

An autoactive NB-LRR gene causes Rht13 dwarfism in wheat.

Semidwarfing genes have greatly increased wheat yields globally, yet the widely used gibberellin (GA)-insensitive genes Rht-B1b and Rht-D1b have disadvantages for seedling emergence. Use of the GA-sensitive semidwarfing gene Rht13 avoids this pleiotropic effect. Here, we show that Rht13 encodes a nucleotide-binding site/leucine-rich repeat (NB-LRR) gene. A point mutation in the semidwarf Rht-B13b allele autoactivates the NB-LRR gene and causes a height reduction comparable with Rht-B1b and Rht-D1b in diverse genetic backgrounds. The autoactive Rht-B13b allele leads to transcriptional up-regulation of pathogenesis-related genes including class III peroxidases associated with cell wall remodeling. Rht13 represents a new class of reduced height (Rht) gene, unlike other Rht genes, which encode components of the GA signaling or metabolic pathways. This discovery opens avenues to use autoactive NB-LRR genes as semidwarfing genes in a range of crop species, and to apply Rht13 in wheat breeding programs using a perfect genetic marker.

Photolysis of caged cytokinin in single cells of Arabidopsis thaliana.

BACKGROUND: Cytokinins are a class of phytohormone that play a crucial role in the development of plants. They are involved in the regulation of nearly every aspect of plant growth, from germination to senescence. The role of cytokinins in many developmental programs is complex and varies both spatially and temporally. Current techniques used to investigate the functions of cytokinins in plant development lack this spatial and temporal resolution required to observe cell-type specific effects. RESULTS: To this end, we present a method of activating a caged cytokinin in single cells. A caged benzyladenine was synthesized, along with caged adenine as a negative control. In vitro testing confirmed ultraviolet light-mediated uncaging, and subsequent root growth assays demonstrated that uncaging produced a cytokinin phenotype. This uncaging was confined to single cells using multiphoton confocal microscopy. Using an Arabidopsis thaliana cytokinin reporter line expressing TCSn::GFP, the resulting GFP expression was confined to the uncaging region, including in single cells. This study presents a novel cell-targeted method of cytokinin delivery, which has the potential to elucidate a broad range of processes in plant development. CONCLUSIONS: We combined multiphoton confocal microscopy and a caged cytokinin treatment, allowing cell type-specific uncaging of a cytokinin in Arabidopsis roots.

Control of root-to-shoot long-distance flow by a key ROS-regulating factor in Arabidopsis.

Inter-tissue communication is instrumental to coordinating the whole-body level behaviour for complex multicellular organisms. However, little is known about the regulation of inter-tissue information exchange. Here we carried out genetic screens for root-to-shoot mobile silencing in Arabidopsis plants with a compromised small RNA-mediated gene silencing movement rate and identified radical-induced cell death 1 (RCD1) as a critical regulator of root-shoot communication. RCD1 belongs to a family of poly (ADP-ribose) polymerase proteins, which are highly conserved across land plants. We found that RCD1 coordinates symplastic and apoplastic movement by modulating the sterol level of lipid rafts. The higher superoxide production in rcd1-knockout plants resulted in lower plasmodesmata (PD) frequency and altered PD structure in the symplasm of the hypocotyl cortex. Furthermore, the mutants showed increased lateral area of tracheary pits, which reduced axial movement. Our study highlights a novel mechanism through which root-to-shoot long-distance signalling can be modulated both symplastically and apoplastically.

Multienvironment QTL analysis delineates a major locus associated with homoeologous exchanges for water-use efficiency and seed yield in canola.

Canola varieties exhibit variation in drought avoidance and drought escape traits, reflecting adaptation to water-deficit environments. Our understanding of underlying genes and their interaction across environments in improving crop productivity is limited. A doubled haploid population was analysed to identify quantitative trait loci (QTL) associated with water-use efficiency (WUE) related traits. High WUE in the vegetative phase was associated with low seed yield. Based on the resequenced parental genome data, we developed sequence-capture-based markers and validated their linkage with carbon isotope discrimination (Δ13 C) in an F2 population. RNA sequencing was performed to determine the expression of candidate genes underlying Δ13 C QTL. QTL contributing to main and QTL × environment interaction effects for Δ13 C and yield were identified. One multiple-trait QTL for Δ13 C, days to flower, plant height, and seed yield was identified on chromosome A09. Interestingly, this QTL region overlapped with a homoeologous exchange (HE) event, suggesting its association with the multiple traits. Transcriptome analysis revealed 121 significantly differentially expressed genes underlying Δ13 C QTL on A09 and C09, including in HE regions. Sorting out the negative relationship between vegetative WUE and seed yield is a priority. Genetic and genomic resources and knowledge so developed could improve canola WUE and yield.

Vascular Connections Into the Grape Berry: The Link of Structural Investment to Seededness.

Vascular bundles in the grape pedicel and berry contain the conduits, phloem and xylem, for transport of water, sugar, nutrients and signals into and through the grape berry and play a critical role in berry growth and composition. Here, we assess the vascular anatomy within the proximal region of the berry. Guided using a 3D berry model generated by micro-CT, differential staining of transverse sections of berries and receptacles was followed by fluorescent microscopy. Morphometric and vascular characteristics were analyzed within the central proximal region (brush zone, a fibrous extension from the pedicel vascular system into the berry) of the seeded cultivars Shiraz and Sauvignon Blanc, as well as the stenospermocarpic cultivars Ruby Seedless and Flame Seedless. Observations revealed a change in vascular arrangement from the receptacle into the berry brush zone and differences in xylem element size as well as xylem and phloem area relationships. Xylem anatomical and derived hydraulic parameters, as well as total tissue area of xylem and phloem varied between cultivars and in receptacle and berry components. Variation in vascular growth between grape pedicels and berries was independent of seededness. Differences in receptacle xylem vessel size and distribution could contribute to cultivar-dependent xylem backflow constraint.

Characterization and Genetic Mapping of Black Root Rot Resistance in Gossypium arboreum L.

Black root rot (BRR) is an economically important disease of cotton and other crops, especially in cooler regions with short growing seasons. Symptoms include black discoloration of the roots, reduced number of lateral roots and stunted or slow plant growth. The cultivated tetraploid Gossypium species are susceptible to BRR. Resistance to BRR was identified in G. arboreum accession BM13H and is associated with reduced and restricted hyphal growth and less sporulation. Transcriptome analysis indicates that BM13H responds to infection at early time points 2- and 3-days post-inoculation, but by day 5, few differentially expressed genes are observed between infected and uninfected roots. Inheritance of BM13H resistance to BRR was evaluated in an F6 recombinant inbred population and shows a single semi-dominant locus conferring resistance that was fine mapped to a region on chromosome 1, containing ten genes including five putative resistance-like genes.

Digging roots is easier with AI.

The scale of root quantification in research is often limited by the time required for sampling, measurement, and processing samples. Recent developments in convolutional neural networks (CNNs) have made faster and more accurate plant image analysis possible, which may significantly reduce the time required for root measurement, but challenges remain in making these methods accessible to researchers without an in-depth knowledge of machine learning. We analyzed root images acquired from three destructive root samplings using the RootPainter CNN software that features an interface for corrective annotation for easier use. Root scans with and without non-root debris were used to test if training a model (i.e. learning from labeled examples) can effectively exclude the debris by comparing the end results with measurements from clean images. Root images acquired from soil profile walls and the cross-section of soil cores were also used for training, and the derived measurements were compared with manual measurements. After 200 min of training on each dataset, significant relationships between manual measurements and RootPainter-derived data were noted for monolith (R2=0.99), profile wall (R2=0.76), and core-break (R2=0.57). The rooting density derived from images with debris was not significantly different from that derived from clean images after processing with RootPainter. Rooting density was also successfully calculated from both profile wall and soil core images, and in each case the gradient of root density with depth was not significantly different from manual counts. Differences in root-length density (RLD) between crops with contrasting root systems were captured using automatic segmentation at soil profiles with high RLD (1-5 cm cm-3) as well with low RLD (0.1-0.3 cm cm-3). Our results demonstrate that the proposed approach using CNN can lead to substantial reductions in root sample processing workloads, increasing the potential scale of future root investigations.

New methods for confocal imaging of infection threads in crop and model legumes.

BACKGROUND: The formation of infection threads in the symbiotic infection of rhizobacteria in legumes is a unique, fascinating, and poorly understood process. Infection threads are tubes of cell wall material that transport rhizobacteria from root hair cells to developing nodules in host roots. They form in a type of reverse tip-growth from an inversion of the root hair cell wall, but the mechanism driving this growth is unknown, and the composition of the thread wall remains unclear. High resolution, 3-dimensional imaging of infection threads, and cell wall component specific labelling, would greatly aid in our understanding of the nature and development of these structures. To date, such imaging has not been done, with infection threads typically imaged by GFP-tagged rhizobia within them, or histochemically in thin sections. RESULTS: We have developed new methods of imaging infection threads using novel and traditional cell wall fluorescent labels, and laser confocal scanning microscopy. We applied a new Periodic Acid Schiff (PAS) stain using rhodamine-123 to the labelling of whole cleared infected roots of Medicago truncatula; which allowed for imaging of infection threads in greater 3D detail than had previously been achieved. By the combination of the above method and a calcofluor-white counter-stain, we also succeeded in labelling infection threads and plant cell walls separately, and have potentially discovered a way in which the infection thread matrix can be visualized. CONCLUSIONS: Our methods have made the imaging and study of infection threads more effective and informative, and present exciting new opportunities for future research in the area.

Phenotypes Conferred by Wheat Multiple Pathogen Resistance Locus, Sr2, Include Cell Death in Response to Biotic and Abiotic Stresses.

The wheat Sr2 locus confers partial resistance to four biotrophic pathogens: wheat stem rust (Puccinia graminis f. sp. tritici), leaf rust (P. triticina), stripe rust (P. striiformis f. sp. tritici), and powdery mildew (Blumeria graminis f. sp. tritici). In addition, Sr2 is linked with a brown coloration of ears and stems, termed pseudo-black chaff (PBC). PBC, initially believed to be elicited by stem rust infection, was subsequently recognized to occur in the absence of pathogen infection. The current study demonstrates that the resistance response to stem rust is associated with the death of photosynthetic cells around rust infection sites in the inoculated leaf sheath. Similarly, Sr2-dependent resistance to powdery mildew was associated with the death of leaf mesophyll cells around mildew infection sites. We demonstrate that PBC occurring in the absence of pathogen inoculation also corresponds with death and the collapse of photosynthetic cells in the affected parts of stems and ears. In addition, Sr2-dependent necrosis was inducible in leaves by application of petroleum jelly or by heat treatments. Thus, Sr2 was found to be associated with cell death, which could be triggered by either biotic or abiotic stresses. Our results suggest a role for the Sr2 locus in controlling cell death in response to stress.

Shootward Movement of CFDA Tracer Loaded in the Bottom Sink Tissues of Arabidopsis.

The symplastic tracer 5(6)-carboxyfluorescein diacetate (CFDA) has been widely applied in living plants to demonstrate the intercellular connection, phloem transport and vascular patterning. This protocol shows bottom-to-top carboxyfluorescein (CF) movement in the Arabidopsis by using the root-cutting and the hypocotyl-pinching procedure respectively. These two different procedures result in different efficiencies of CF movement: about 91% appearance of CF in the shoots with the hypocotyl-pinching procedure, whereas only about 70% appearance of CF with the root-cutting procedure. The simple change of loading sites, resulting in significant changes in the mobile efficiency of this symplastic dye, suggests CF movement might be subject to the symplastic regulation, most probably by the root-hypocotyl junction.

Response of plasmodesmata formation in leaves of C4 grasses to growth irradiance.

Rapid metabolite diffusion across the mesophyll (M) and bundle sheath (BS) cell interface in C4 leaves is a key requirement for C4 photosynthesis and occurs via plasmodesmata (PD). Here, we investigated how growth irradiance affects PD density between M and BS cells and between M cells in two C4 species using our PD quantification method, which combines three-dimensional laser confocal fluorescence microscopy and scanning electron microscopy. The response of leaf anatomy and physiology of NADP-ME species, Setaria viridis and Zea mays to growth under different irradiances, low light (100 µmol m-2  s-1 ), and high light (1,000 µmol m-2  s-1 ), was observed both at seedling and established growth stages. We found that the effect of growth irradiance on C4 leaf PD density depended on plant age and species. The high light treatment resulted in two to four-fold greater PD density per unit leaf area than at low light, due to greater area of PD clusters and greater PD size in high light plants. These results along with our finding that the effect of light on M-BS PD density was not tightly linked to photosynthetic capacity suggest a complex mechanism underlying the dynamic response of C4 leaf PD formation to growth irradiance.

3D Clearing and Molecular Labeling in Plant Tissues.

Plant histology and imaging traditionally involve the transformation of tissues into thin sections to minimize light scatter in opaque material, allowing optical clarity and high-resolution microscopy. Recently, new techniques in 3D tissue clearing, including PEA-CLARITY, have been developed to minimize light scatter within intact, whole samples. These techniques can achieve equivalent microscopic resolution to that of thin section imaging with the added benefit of maintaining the original 3D structure and position of biomolecules of interest. Furthermore, PEA-CLARITY is compatible with standard stains and immunohistochemistry, allowing molecular interrogation of intact, 3D tissues. This chapter outlines the current methods available for 3D histology in plants and details the materials, equipment, reagents, and procedure for the PEA-CLARITY technique.

Altered Expression of the Malate-Permeable Anion Channel OsALMT4 Reduces the Growth of Rice Under Low Radiance.

We examined the function of OsALMT4 in rice (Oryza sativa L.) which is a member of the aluminum-activated malate transporter family. Previous studies showed that OsALMT4 localizes to the plasma membrane and that expression in transgenic rice lines results in a constitutive release of malate from the roots. Here, we show that OsALMT4 is expressed widely in roots, shoots, flowers, and grain but not guard cells. Expression was also affected by ionic and osmotic stress, light and to the hormones ABA, IAA, and salicylic acid. Malate efflux from the transgenic plants over-expressing OsALMT4 was inhibited by niflumate and salicylic acid. Growth of transgenic lines with either increased OsALMT4 expression or reduced expression was measured in different environments. Light intensity caused significant differences in growth between the transgenic lines and controls. When day-time light was reduced from 700 to 300 µmol m-2s-1 independent transgenic lines with either increased or decreased OsALMT4 expression accumulated less biomass compared to their null controls. This response was not associated with differences in photosynthetic capacity, stomatal conductance or sugar concentrations in tissues. We propose that by disrupting malate fluxes across the plasma membrane carbon partitioning and perhaps signaling are affected which compromises growth under low light. We conclude that OsALMT4 is expressed widely in rice and facilitates malate efflux from different cell types. Altering OsALMT4 expression compromises growth in low-light environments.

Multiple mechanisms for enhanced plasmodesmata density in disparate subtypes of C4 grasses.

Proliferation of plasmodesmata (PD) connections between bundle sheath (BS) and mesophyll (M) cells has been proposed as a key step in the evolution of two-cell C4 photosynthesis; However, a lack of quantitative data has hampered further exploration and validation of this hypothesis. In this study, we quantified leaf anatomical traits associated with metabolite transport in 18 species of BEP and PACMAD grasses encompassing four origins of C4 photosynthesis and all three C4 subtypes (NADP-ME, NAD-ME, and PCK). We demonstrate that C4 leaves have greater PD density between M and BS cells than C3 leaves. We show that this greater PD density is achieved by increasing either the pit field (cluster of PD) area or the number of PD per pit field area. NAD-ME species had greater pit field area per M-BS interface than NADP-ME or PCK species. In contrast, NADP-ME and PCK species had lower pit field area with increased number of PD per pit field area than NAD-ME species. Overall, PD density per M-BS cell interface was greatest in NAD-ME species while PD density in PCK species exhibited the largest variability. Finally, the only other anatomical characteristic that clearly distinguished C4 from C3 species was their greater Sb value, the BS surface area to subtending leaf area ratio. In contrast, BS cell volume was comparable between the C3 and C4 grass species examined.

Nodulating Legumes Are Distinguished by a Sensitivity to Cytokinin in the Root Cortex Leading to Pseudonodule Development.

Root nodule symbiosis (RNS) is a feature confined to a single clade of plants, the Fabids. Among Fabids capable of RNS, legumes form root cortex-based nodules in symbioses with rhizobia, while actinorhizal species form lateral root-based nodules with actinomycetes. Cytokinin has previously been shown to be sufficient for "pseudonodule" initiation in model legumes. Here, we tested whether this response correlates with the ability to nodulate across a range of plant species. We analyzed the formation of pseudonodules in 17 nodulating and non-nodulating legume species, and 11 non-legumes, including nodulating actinorhizal species, using light and fluorescence microscopy. Cytokinin-induced pseudonodules arising from cortical cell divisions occurred in all nodulating legume species, but not in any of the other species, including non-nodulating legumes. Pseudonodule formation was dependent on the CRE1 cytokinin receptor in Medicago truncatula. Inhibition of root growth by cytokinin occurred across plant groups, indicating that pseudonodule development is the result of a specific cortical cytokinin response unique to nodulating legumes. Lack of a cortical cytokinin response from the Arabidopsis thaliana cytokinin reporter TCSn::GFP supported this hypothesis. Our results suggest that the ability to form cortical cell-derived nodules was gained in nodulating legumes, and likely lost in non-nodulating legumes, due to a specific root cortical response to cytokinin.

Comparative Lipidomics and Proteomics of Lipid Droplets in the Mesocarp and Seed Tissues of Chinese Tallow (Triadica sebifera).

Lipid droplets (LDs) are composed of a monolayer of phospholipids (PLs), surrounding a core of non-polar lipids that consist mostly of triacylglycerols (TAGs) and to a lesser extent diacylglycerols. In this study, lipidome analysis illustrated striking differences in non-polar lipids and PL species between LDs derived from Triadica sebifera seed kernels and mesocarp. In mesocarp LDs, the most abundant species of TAG contained one C18:1 and two C16:0 and fatty acids, while TAGs containing three C18 fatty acids with higher level of unsaturation were dominant in the seed kernel LDs. This reflects the distinct differences in fatty acid composition of mesocarp (palmitate-rich) and seed-derived oil (α-linoleneate-rich) in T. sebifera. Major PLs in seed LDs were found to be rich in polyunsaturated fatty acids, in contrast to those with relatively shorter carbon chain and lower level of unsaturation in mesocarp LDs. The LD proteome analysis in T. sebifera identified 207 proteins from mesocarp, and 54 proteins from seed kernel, which belong to various functional classes including lipid metabolism, transcription and translation, trafficking and transport, cytoskeleton, chaperones, and signal transduction. Oleosin and lipid droplets associated proteins (LDAP) were found to be the predominant proteins associated with LDs in seed and mesocarp tissues, respectively. We also show that LDs appear to be in close proximity to a number of organelles including the endoplasmic reticulum, mitochondria, peroxisomes, and Golgi apparatus. This comparative study between seed and mesocarp LDs may shed some light on the structure of plant LDs and improve our understanding of their functionality and cellular metabolic networks in oleaginous plant tissues.

Genetic enhancement of oil content in potato tuber (Solanum tuberosum L.) through an integrated metabolic engineering strategy.

Potato tuber is a high yielding food crop known for its high levels of starch accumulation but only negligible levels of triacylglycerol (TAG). In this study, we evaluated the potential for lipid production in potato tubers by simultaneously introducing three transgenes, including WRINKLED 1 (WRI1), DIACYLGLYCEROL ACYLTRANSFERASE 1 (DGAT1) and OLEOSIN under the transcriptional control of tuber-specific (patatin) and constitutive (CaMV-35S) promoters. This coordinated metabolic engineering approach resulted in over a 100-fold increase in TAG accumulation to levels up to 3.3% of tuber dry weight (DW). Phospholipids and galactolipids were also found to be significantly increased in the potato tuber. The increase of lipids in these transgenic tubers was accompanied by a significant reduction in starch content and an increase in soluble sugars. Microscopic examination revealed that starch granules in the transgenic tubers had more irregular shapes and surface indentations when compared with the relatively smooth surfaces of wild-type starch granules. Ultrastructural examination of lipid droplets showed their close proximity to endoplasmic reticulum and mitochondria, which may indicate a dynamic interaction with these organelles during the processes of lipid biosynthesis and turnover. Increases in lipid levels were also observed in the transgenic potato leaves, likely due to the constitutive expression of DGAT1 and incomplete tuber specificity of the patatin promoter. This study represents an important proof-of-concept demonstration of oil increase in tubers and provides a model system to further study carbon reallocation during development of nonphotosynthetic underground storage organs.

Step changes in leaf oil accumulation via iterative metabolic engineering.

Synthesis and accumulation of plant oils in the entire vegetative biomass offers the potential to deliver yields surpassing those of oilseed crops. However, current levels still fall well short of those typically found in oilseeds. Here we show how transcriptome and biochemical analyses pointed to a futile cycle in a previously established Nicotiana tabacum line, accumulating up to 15% (dry weight) of the storage lipid triacylglycerol in leaf tissue. To overcome this metabolic bottleneck, we either silenced the SDP1 lipase or overexpressed the Arabidopsis thaliana LEC2 transcription factor in this transgenic background. Both strategies independently resulted in the accumulation of 30-33% triacylglycerol in leaf tissues. Our results demonstrate that the combined optimization of de novo fatty acid biosynthesis, storage lipid assembly and lipid turnover in leaf tissue results in a major overhaul of the plant central carbon allocation and lipid metabolism. The resulting further step changes in oil accumulation in the entire plant biomass offers the possibility of delivering yields that outperform current oilseed crops.

Quantifying the plant actin cytoskeleton response to applied pressure using nanoindentation.

Detection of potentially pathogenic microbes through recognition by plants and animals of both physical and chemical signals associated with the pathogens is vital for host well-being. Signal perception leads to the induction of a variety of responses that augment pre-existing, constitutive defences. The plant cell wall is a highly effective preformed barrier which becomes locally reinforced at the infection site through delivery of new wall material by the actin cytoskeleton. Although mechanical stimulation can produce a reaction, there is little understanding of the nature of physical factors capable of triggering plant defence. Neither the magnitude of forces nor the contact time required has been quantified. In the study reported here, mechanical stimulation with a tungsten microneedle has been used to quantify the response of Arabidopsis plants expressing an actin-binding protein tagged with green fluorescent protein (GFP) to reveal the organisation of the actin cytoskeleton. Using confocal microscopy, the response time for actin reorganisation in epidermal cells of Arabidopsis hypocotyls was shown to be 116 ± 49 s. Using nanoindentation and a diamond spherical tip indenter, the magnitude of the forces capable of triggering an actin response has been quantified. We show that Arabidopsis hypocotyl cells can detect a force as small as 4 µN applied for as short a time as 21.6 s to trigger reorganisation of the actin cytoskeleton. This force is an order of magnitude less than the potential invasive force determined for a range of fungal and oomycete plant pathogens. To our knowledge, this is the first quantification of the magnitude and duration of mechanical forces capable of stimulating a structural defence response in a plant cell.