Physiological and Proteomic Analyses of Different Ecotypes of Reed (Phragmites communis) in Adaption to Natural Drought and Salinity

Drought and salinity are the two major abiotic stresses constraining the crop yield worldwide. Both of them trigger cellular dehydration and cause osmotic stress which leads to cytosolic and vacuolar volume reduction. However, whether plants share a similar tolerance mechanism in response to these two stresses under natural conditions has seldom been comparatively reported. There are three different ecotypes of reed within a 5 km2 region in the Badanjilin desert of Northwest China. Taking the typical swamp reed (SR) as a control, we performed a comparative study on the adaption mechanisms of the two terrestrial ecotypes: dune reed (DR) and heavy salt meadow reed (HSMR) by physiological and proteomic approaches coupled with bioinformatic analysis. The results showed that HSMR and DR have evolved C4-like photosynthetic and anatomical characteristics, such as the increased bundle sheath cells (BSCs) and chloroplasts in BSCs, higher density of veins, and lower density and aperture of stomata. In addition, the thylakoid membrane fluidity also plays an important role in their higher drought and salinity tolerance capability. The proteomic results further demonstrated that HSMR and DR facilitated the regulation of proteins associated with photosynthesis and energy metabolism, lipid metabolism, transcription and translation, and stress responses to well-adapt to the drought and salinity conditions. Overall, our results demonstrated that HSMR and DR shaped a similar adaption strategy from the structural and physiological levels to the molecular scale to ensure functionality in a harsh environment.

Leaf Epidermis: The Ambiguous Symplastic Domain

The ability to develop secondary (post-cytokinetic) plasmodesmata (PD) is an important evolutionary advantage that helps in creating symplastic domains within the plant body. Developmental regulation of secondary PD formation is not completely understood. In flowering plants, secondary PD occur exclusively between cells from different lineages, e.g., at the L1/L2 interface within shoot apices, or between leaf epidermis (L1-derivative), and mesophyll (L2-derivative). However, the highest numbers of secondary PD occur in the minor veins of leaf between bundle sheath cells and phloem companion cells in a group of plant species designated “symplastic” phloem loaders, as opposed to “apoplastic” loaders. This poses a question of whether secondary PD formation is upregulated in general in symplastic loaders. Distribution of PD in leaves and in shoot apices of two symplastic phloem loaders, Alonsoa meridionalis and Asarina barclaiana, was compared with that in two apoplastic loaders, Solanum tuberosum (potato) and Hordeum vulgare (barley), using immunolabeling of the PD-specific proteins and transmission electron microscopy (TEM), respectively. Single-cell sampling was performed to correlate sugar allocation between leaf epidermis and mesophyll to PD abundance. Although the distribution of PD in the leaf lamina (except within the vascular tissues) and in the meristem layers was similar in all species examined, far fewer PD were found at the epidermis/epidermis and mesophyll/epidermis boundaries in apoplastic loaders compared to symplastic loaders. In the latter, the leaf epidermis accumulated sugar, suggesting sugar import from the mesophyll via PD. Thus, leaf epidermis and mesophyll might represent a single symplastic domain in Alonsoa meridionalis and Asarina barclaiana.

ZmFdC2 Encoding a Ferredoxin Protein With C-Terminus Extension Is Indispensable for Maize Growth

As important electron carriers, ferredoxin (Fd) proteins play important roles in photosynthesis, and the assimilation of CO2, nitrate, sulfate, and other metabolites. In addition to the well-studied Fds, plant genome encodes two Fd-like protein members named FdC1 and FdC2, which have extension regions at the C-terminus of the 2Fe-2S cluster. Mutation or overexpression of FdC genes caused alterations in photosynthetic electron transfer rate in rice and Arabidopsis. Maize genome contains one copy of each FdC gene. However, the functions of these genes have not been reported. In this study, we identified the ZmFdC2 gene by forward genetics approach. Mutation of this gene causes impaired photosynthetic electron transport and collapsed chloroplasts. The mutant plant is seedling-lethal, indicating the indispensable function of ZmFdC2 gene in maize development. The ZmFdC2 gene is specifically expressed in photosynthetic tissues and induced by light treatment, and the encoded protein is localized on chloroplast, implying its specialized function in photosynthesis. Furthermore, ZmFdC2 expression was detected in both mesophyll cells and bundle sheath cells, the two cell types specialized for C4 and C3 photosynthesis pathways in maize. Epigenomic analyses showed that ZmFdC2 locus was enriched for active histone modifications. Our results demonstrate that ZmFdC2 is a key component of the photosynthesis pathway and is crucial for the development of maize.

Formation of Proto-Kranz in C3 Rice Induced by Spike-Stalk Injection Method

Introduction of C4 photosynthetic traits into C3 crops is an important strategy for improving photosynthetic capacity and productivity. Here, we report the research results of a variant line of sorghum–rice (SR) plant with big panicle and high spikelet density by introducing sorghum genome DNA into rice by spike-stalk injection. The whole-genome resequencing showed that a few sorghum genes could be integrated into the rice genome. Gene expression was confirmed for two C4 photosynthetic enzymes containing pyruvate, orthophosphate dikinase and phosphoenolpyruvate carboxykinase. Exogenous sorghum DNA integration induced a series of key traits associated with the C4 pathway called “proto-Kranz” anatomy, including leaf thickness, bundle sheath number and size, and chloroplast size in bundle sheath cells. Significantly, transgenic plants exhibited enhanced photosynthetic capacity resulting from both photosynthetic CO2-concentrating effect and improved energy balance, which led to an increase in carbohydrate levels and productivity. Furthermore, such rice plant exhibited delayed leaf senescence. In summary, this study provides a proof for the feasibility of inducing the transition from C3 leaf anatomy to proto-Kranz by spike-stalk injection to achieve efficient photosynthesis and increase productivity.

The bundle sheath of rice is conditioned to play an active role in water transport as well as sulfur assimilation and jasmonic acid synthesis

Leaves comprise multiple cell types but our knowledge of the patterns of gene expression that underpin their functional specialization is fragmentary. Our understanding and ability to undertake rational redesign of these cells is therefore limited. We aimed to identify genes associated with the incompletely understood bundle sheath of C3 plants, which represents a key target associated with engineering traits such as C4 photosynthesis into rice. To better understand veins, bundle sheath and mesophyll cells of rice we used laser capture microdissection followed by deep sequencing. Gene expression of the mesophyll is conditioned to allow coenzyme metabolism and redox homeostasis as well as photosynthesis. In contrast, the bundle sheath is specialized in water transport, sulphur assimilation and jasmonic acid biosynthesis. Despite the small chloroplast compartment of bundle sheath cells, substantial photosynthesis gene expression was detected. These patterns of gene expression were not associated with presence/absence of particular transcription factors in each cell type, but rather gradients in expression across the leaf. Comparative analysis with C3Arabidopsis identified a small gene-set preferentially expressed in bundle sheath cells of both species. This included genes encoding transcription factors from fourteen orthogroups, and proteins allowing water transport, sulphate assimilation and jasmonic acid synthesis. The most parsimonious explanation for our findings is that bundle sheath cells from the last common ancestor of rice and Arabidopsis was specialized in this manner, and since the species diverged these patterns of gene expression have been maintained. Significance statement The role of bundle sheath cells in C4 species have been studied intensively but this is not the case in leaves that use the ancestral C3 pathway. Here, we show that gene expression in the bundle sheath of rice is specialized to allow sulphate and nitrate reduction, water transport and jasmonate synthesis, and comparative analysis with Arabidopsis indicates ancient roles for bundle sheath cells in water transport, sulphur and jasmonate synthesis.

Stress is basic: ABA alkalinizes both the xylem sap and the cytosol of Arabidopsis vascular bundle sheath cells by inhibiting their P-type H+-ATPase and stimulating their V-type H+-ATPase

ABSTRACTBACKGROUND AND HYPOTHESIS•Under water deprivation, in many perennial species, the stress hormone, ABA, appears in the xylem sap in the shoot (including leaf) veins and the xylem sap pH (pHEXT) increases. This study aimed to test the hypothesis that ABA is the signal for an altered proton balance of the leaf-vein-enwrapping bundle sheath cells (BSCs).METHODS•Plant Material. We used a few Arabidopsis thaliana (L.) Heynh. genotypes: wildtype (WT) of two accessions, Landsberg erecta (Ler) and Columbia (Col), and a few mutants and transformants in these backgrounds.•H+-Pumps activities. We monitored ABA effects on the H+-pump activities in the BSCs cytosol-delimiting membranes (plasma membrane and tonoplast) by monitoring the cytosol and the xylem pH, and the membrane potential (EM), by imaging the fluorescence of pH- and membrane potential (EM)-reporting probes: (a) the BSCs’ pHEXT – with the ratiometric fluorescent dye FITC-dextran petiole-fed into detached leaves in unbuffered xylem perfusion solution (XPS), (b) the BSCs’ pHCYT – with the ratiometric dye SNARF1 loaded into BSCs isolated protoplasts, and (c) the BSCs’ EM – with the ratiometric dye di- 8-ANEPPS.RESULTS•ABA increased the pHEXT; this response was abolished in an abi1-1 mutant with impaired signaling via a PP2C (ABI1) and in an aha2-4 mutant with knocked-down AHA2;•ABA depolarized the WT BSCs;•ABA increased pHCYT irrespective of AHA2 activity (i.e., whether or not AHA was inhibited by vanadate, or in the aha2-4 mutant);•The ABA-induced cytosol alkalinization was abolished in the absence of VHA activity (i.e., when VHA was inhibited by bafilomycin A1, or in the vha-a2 vha-a3 double mutant with inactive VHA);•All these results resemble the ABA effect on GCs;•In contrast to GCs, AHA2 and not AHA1 is the ABA major target in BSCs;•Blue light (BL) enabled the response of the BSCs’ VHA to ABA;•The ABA- and BL-signaling pathways acting on both BSCs’ pumps, AHA2 and VHA, are likely to be BSCs autonomous, based on (a) the presence in the BSCs of many genes of the ABA- and BL-signaling pathways and (b) ABA responses (depolarization and pHCYT elevation) demonstrated under BL in isolated protoplasts.SIGNIFICANCE STATEMENTWe reveal here an alkalinizing effect of the plant drought-stress hormone ABA on the pH on both sides of the plasmalemma of the vein-enwrapping bundle sheath cells (BSCs), due to ABA inhibition of the BSCs’ AHA2, the plasmalemma H+- ATPase and stimulation of VHA, their vacuolar H+-ATPase. Since pH affects the BSCs’ selective regulation of solute and water fluxes into the leaf, these H+- pumps may be attractive targets for manipulations aiming to improve plant drought response.

Understanding C4 photosynthesis in Setaria by a proteomic and kinetic approach

Plants performing C4 photosynthesis have a higher productivity per crop area related to an optimized use of water and nutrients. This is achieved through a series of anatomical and biochemical features that allow the concentration of CO2 around RuBisCO. In C4 plants the photosynthetic reactions are distributed between two cell types, they initially fix the carbon to C4 acids within the mesophyll cells (M) and then transport these compounds to the bundle sheath cells (BS), where they are decarboxylated so that the resulting CO2 is incorporated into the Calvin cycle (CC). This work is focused on the comparative analysis of the proteins present in M and BS of Setaria viridis, a C4 model close relative of several major feed, fuel, and bioenergy grasses. The integration of kinetic and proteomic approaches agrees that the C4 compound malate is mainly decarboxylated in the chloroplasts of BS cells by NADP-malic enzyme (NADP-ME). Besides, NAD-malic enzyme (NAD-ME) located in the mitochondria could also contribute to the C4 carbon shuttle. We presented evidence of metabolic strategies that involve chloroplastic, mitochondrial and peroxisomal proteins to avoid the leakage of C4 intermediates in order to sustain an efficient photosynthetic performance.

Flagellin triggers mesophyll dehydration: An early PTI defense against bacterial establishment in intercellular spaces

Plants have evolved various mechanisms to defend themselves against pathogens. Many pathogens induce the formation of water-soaked lesions during early infection under conditions of high atmospheric humidity. These water-soaked spots are caused by the disruption of the plasma membrane or cell wall integrity due to various activities of effector proteins during infection. We hypothesized that bacterial PAMP-flagellin plays a role in modulating the cell-membrane permeability that controls the availability of water in the apoplast, to prevent bacterial establishment on the cell wall during the early stages of the PAMP-triggered immunity (PTI) response. Our results revealed that the conductivity of hydraulic pathways in the leaf was reduced in response to flagellin22 (flg22). The cellular osmotic water permeability (Pf) of both mesophyll cells and bundle-sheath cells was dramatically reduced in response to flg22 treatment. Moreover, the whole-leaf hydraulic conductance (Kleaf) was also reduced in response to flg22 treatment. The fact that the Pf of mesophyll cells of an aquaporin (AQP) mutant was not affected by the flg22 treatment suggests the involvement of AQP channels in the flg22-induced Pf reduction signal transduction pathway. We conclude that the binding of flagellin to their receptors elicits signals to close AQPs, consequently reducing the water content of the cell wall and intercellular spaces and leading to a more negative water potential. This serves as an early PTI response to pathogen attack, which, in turn, might decrease the rate of bacterial growth and establishment in the apoplast.

Transmission of Engineered Plastids in Sugarcane, a C4 Monocotyledonous Plant, Reveals that Sorting of Preprogrammed Progenitor Cells Produce Heteroplasmy

We report here plastid transformation in sugarcane using biolistic transformation and embryogenesis-based regeneration approaches. Somatic embryos were developed from unfurled leaf sections, containing preprogrammed progenitor cells, to recover transformation events on antibiotic-containing regeneration medium. After developing a proficient regeneration system, the FLARE-S (fluorescent antibiotic resistance enzyme, spectinomycin and streptomycin) expression cassette that carries species-specific homologous sequence tails was used to transform plastids and track gene transmission and expression in sugarcane. Plants regenerated from streptomycin-resistant and genetically confirmed shoots were subjected to visual detection of the fluorescent enzyme using a fluorescent stereomicroscope, after genetic confirmation. The resultant heteroplasmic shoots remained to segregate on streptomycin-containing MS medium, referring to the unique pattern of division and sorting of cells in C4 monocotyledonous compared to C3 monocotyledonous and dicotyledonous plants since in sugarcane bundle sheath and mesophyll cells are distinct and sort independently after division. Hence, the transformation of either mesophyll or bundle sheath cells will develop heteroplasmic transgenic plants, suggesting the transformation of both types of cells. Whilst developed transgenic sugarcane plants are heteroplasmic, and selection-based regeneration protocol envisaging the role of division and sorting of cells in the purification of transplastomic demands further improvement, the study has established many parameters that may open up exciting possibilities to express genes of agricultural or pharmaceutical importance in sugarcane.