Kosala Ranathunge

Defective Pollen Wall Is Required for Anther and Microspore Development in Rice and Encodes a Fatty Acyl Carrier Protein Reductase

A rice male-sterile mutant, defective pollen wall (dpw), which has defective anthers and pollen grains, is isolated and characterized. DPW is found to encode a fatty acyl carrier protein reductase that is active during the synthesis of the anther cuticle and pollen sporopollenin. Aliphatic alcohols naturally exist in many organisms as important cellular components; however, their roles in extracellular polymer biosynthesis are poorly defined. We report here the isolation and characterization of a rice (Oryza sativa) male-sterile mutant, defective pollen wall (dpw), which displays defective anther development and degenerated pollen grains with an irregular exine. Chemical analysis revealed that dpw anthers had a dramatic reduction in cutin monomers and an altered composition of cuticular wax, as well as soluble fatty acids and alcohols. Using map-based cloning, we identified the DPW gene, which is expressed in both tapetal cells and microspores during anther development. Biochemical analysis of the recombinant DPW enzyme shows that it is a novel fatty acid reductase that produces 1-hexadecanol and exhibits >270-fold higher specificity for palmiltoyl-acyl carrier protein than for C16:0 CoA substrates. DPW was predominantly targeted to plastids mediated by its N-terminal transit peptide. Moreover, we demonstrate that the monocot DPW from rice complements the dicot Arabidopsis thaliana male sterile2 (ms2) mutant and is the probable ortholog of MS2. These data suggest that DPWs participate in a conserved step in primary fatty alcohol synthesis for anther cuticle and pollen sporopollenin biosynthesis in monocots and dicots.

Suberin research in the genomics era--new interest for an old polymer.

Suberin is an apoplastic biopolymer with tissue-specific deposition in the cell walls of the endo- and exodermis of roots, of periderms including wound periderm and other border tissues. Suberised cell walls contain both polyaliphatic and polyaromatic domains which are supposedly cross-linked. The predominant aliphatic components are ω-hydroxyacids, α,ω-diacids, fatty acids and primary alcohols, whereas hydroxycinnamic acids, especially ferulic acid, are the main components of the polyaromatic domain. Although the monomeric composition of suberin has been known for decades, its biosynthesis and deposition has mainly been a subject of speculation. Only recently, significant progress elucidating suberin biosynthesis has been achieved using molecular genetic approaches, especially in the model species Arabidopsis. In parallel, the long-standing hypothesis that suberin functions as an apoplastic barrier has been corroborated by sophisticated, quantitative physiological studies in the past decade. These studies demonstrated that suberised cell walls could act as barriers, minimising the movement of water and nutrients, restricting pathogen invasion and impeding toxic gas diffusion. In addition, suberised cell walls provide a barrier to radial oxygen loss from roots to the anaerobic root substrate in wetland plants. The recent onset of multidisciplinary approaches combining genetic, analytical and physiological studies has begun to deliver further insights into the physiological importance of suberin depositions in plants.

Root apoplastic barriers block Na+ transport to shoots in rice (Oryza sativa L.)

Rice is an important crop that is very sensitive to salinity. However, some varieties differ greatly in this feature, making investigations of salinity tolerance mechanisms possible. The cultivar Pokkali is salinity tolerant and is known to have more extensive hydrophobic barriers in its roots than does IR20, a more sensitive cultivar. These barriers located in the root endodermis and exodermis prevent the direct entry of external fluid into the stele. However, it is known that in the case of rice, these barriers are bypassed by most of the Na+ that enters the shoot. Exposing plants to a moderate stress of 100 mM NaCl resulted in deposition of additional hydrophobic aliphatic suberin in both cultivars. The present study demonstrated that Pokkali roots have a lower permeability to water (measured using a pressure chamber) than those of IR20. Conditioning plants with 100 mM NaCl effectively reduced Na+ accumulation in the shoot and improved survival of the plants when they were subsequently subjected to a lethal stress of 200 mM NaCl. The Na+ accumulated during the conditioning period was rapidly released when the plants were returned to the control medium. It has been suggested that the location of the bypass flow is around young lateral roots, the early development of which disrupts the continuity of the endodermal and exodermal Casparian bands. However, in the present study, the observed increase in lateral root densities during stress in both cultivars did not correlate with bypass flow. Overall the data suggest that in rice roots Na+ bypass flow is reduced by the deposition of apoplastic barriers, leading to improved plant survival under salt stress.

ABCG transporters are required for suberin and pollen wall extracellular barriers in Arabidopsis.

Plants make tough barriers outside their cells in precise anatomical locations to control water and solute movement through their tissues. This work demonstrates that a family of transport proteins is required in Arabidopsis to form such barriers in roots and in seed coats and to make a functional pollen wall. Effective regulation of water balance in plants requires localized extracellular barriers that control water and solute movement. We describe a clade of five Arabidopsis thaliana ABCG half-transporters that are required for synthesis of an effective suberin barrier in roots and seed coats (ABCG2, ABCG6, and ABCG20) and for synthesis of an intact pollen wall (ABCG1 and ABCG16). Seed coats of abcg2 abcg6 abcg20 triple mutant plants had increased permeability to tetrazolium red and decreased suberin content. The root system of triple mutant plants was more permeable to water and salts in a zone complementary to that affected by the Casparian strip. Suberin of mutant roots and seed coats had distorted lamellar structure and reduced proportions of aliphatic components. Root wax from the mutant was deficient in alkylhydroxycinnamate esters. These mutant plants also had few lateral roots and precocious secondary growth in primary roots. abcg1 abcg16 double mutants defective in the other two members of the clade had pollen with defects in the nexine layer of the tapetum-derived exine pollen wall and in the pollen-derived intine layer. Mutant pollen collapsed at the time of anther desiccation. These mutants reveal transport requirements for barrier synthesis as well as physiological and developmental consequences of barrier deficiency.

Water and solute permeabilities of Arabidopsis roots in relation to the amount and composition of aliphatic suberin

Although it is implied that suberized apoplastic barriers of roots negatively correlate with water and solute permeabilities, direct transport measurements across roots with altered amounts and compositions of aliphatic suberin are scarce. In the present study, hydroponically grown Arabidopsis wild types (Col8 and Col0) and different suberin mutants with altered amounts and/or compositions (horst, esb1-1, and esb1-2) were used to test this hypothesis. Detailed histochemical studies revealed late development of Casparian bands and suberin lamellae in the horst mutant compared with wild types and esb mutants. Suberin analysis with gas chromatography and mass spectrometry (GC-MS) showed that the horst mutant had ∼33% lower amounts of aliphatic monomers than Col8 and Col0. In contrast, enhanced suberin mutants (esb1-1 and esb1-2) had twice the amount of suberin as the wild types. Correlative permeability measurements, which were carried out for the first time with a root pressure probe for Arabidopsis, revealed that the hydraulic conductivity (Lpr) and NaCl permeability (Psr) of the whole root system of the horst mutant were markedly greater than in the respective wild types. This was reflected by the total amounts of aliphatic suberin determined in the roots. However, increased levels of aliphatic suberin in esb mutants failed to reduce either water or NaCl permeabilities below those of the wild types. It was concluded that the simple view and the conventional assumption that the amount of root suberin negatively correlates with permeability may not always be true. The aliphatic monomer arrangement in the suberin biopolymer and its microstructure also play a role in apoplastic barrier formation.

AMT1;1 transgenic rice plants with enhanced NH4 + permeability show superior growth and higher yield under optimal and suboptimal NH4 + conditions

The major source of nitrogen for rice (Oryza sativa L.) is ammonium (NH4 +). The NH4 + uptake of roots is mainly governed by membrane transporters, with OsAMT1;1 being a prominent member of the OsAMT1 gene family that is known to be involved in NH4 + transport in rice plants. However, little is known about its involvement in NH4 + uptake in rice roots and subsequent effects on NH4 + assimilation. This study shows that OsAMT1;1 is a constitutively expressed, nitrogen-responsive gene, and its protein product is localized in the plasma membrane. Its expression level is under the control of circadian rhythm. Transgenic rice lines (L-2 and L-3) overexpressing the OsAMT1;1 gene had the same root structure as the wild type (WT). However, they had 2-fold greater NH4 + permeability than the WT, whereas OsAMT1;1 gene expression was 20-fold higher than in the WT. Analogous to the expression, transgenic lines had a higher NH4 + content in the shoots and roots than the WT. Direct NH4 + fluxes in the xylem showed that the transgenic lines had significantly greater uptake rates than the WT. Higher NH4 + contents also promoted higher expression levels of genes in the nitrogen assimilation pathway, resulting in greater nitrogen assimilates, chlorophyll, starch, sugars, and grain yield in transgenic lines than in the WT under suboptimal and optimal nitrogen conditions. OsAMT1;1 also enhanced overall plant growth, especially under suboptimal NH4 + levels. These results suggest that OsAMT1;1 has the potential for improving nitrogen use efficiency, plant growth, and grain yield under both suboptimal and optimal nitrogen fertilizer conditions.

The Rice R2R3-MYB Transcription Factor OsMYB55 Is Involved in the Tolerance to High Temperature and Modulates Amino Acid Metabolism

Temperatures higher than the optimum negatively affects plant growth and development. Tolerance to high temperature is a complex process that involves several pathways. Understanding this process, especially in crops such as rice, is essential to prepare for predicted climate changes due to global warming. Here, we show that OsMYB55 is induced by high temperature and overexpression of OsMYB55 resulted in improved plant growth under high temperature and decreased the negative effect of high temperature on grain yield. Transcriptome analysis revealed an increase in expression of several genes involved in amino acids metabolism. We demonstrate that OsMYB55 binds to the promoter regions of target genes and directly activates expression of some of those genes including glutamine synthetase (OsGS1;2) glutamine amidotransferase (GAT1) and glutamate decarboxylase 3 (GAD3). OsMYB55 overexpression resulted in an increase in total amino acid content and of the individual amino acids produced by the activation of the above mentioned genes and known for their roles in stress tolerance, namely L-glutamic acid, GABA and arginine especially under high temperature condition. In conclusion, overexpression of OsMYB55 improves rice plant tolerance to high temperature, and this high tolerance is associated with enhanced amino acid metabolism through transcription activation.

Wax Crystal-Sparse Leaf1 encodes a β–ketoacyl CoA synthase involved in biosynthesis of cuticular waxes on rice leaf

Cuticular waxes, forming the plant/atmosphere interface of plants colonizing the terrestrial environment, are complex mixtures of very-long chain fatty acids (VLCFAs) and their derivatives. In VLCFAs biosynthesis, β-ketoacyl CoA synthase (E.C.2.3.1.119, KCS) is the key enzyme. Using T-DNA insertional mutagenesis, we identified a cuticle-deficient rice mutant, which displayed a pleiotropic phenotype including reduced growth, leaf fusion, sparse wax crystals, enhanced sensitivity to drought and low fertility. Further analysis indicated that T-DNA was inserted in the 5′-UTR intron of the affected gene, Wax Crystal-Sparse Leaf1 (WSL1), and abnormal transcript caused the loss-of-function of WSL1 gene. Genetic complementation experiment confirmed the function of the candidate gene. WSL1 was predicted to encode a polypeptide containing a conserved FAE1_CUT1_RppA domain typical of the KCS family proteins. Qualitative and quantitative wax composition analyses by gas chromatography–mass spectrometry (GC–MS) demonstrated a marked reduction of total cuticular wax load on wsl1 leaf blades and sheaths, and VLCFA precursors of C20–C24 decreased in both. Moreover, ubiquitous expression of the WSL1 gene gave a hint that WSL1-catalyzed elongation of VLCFAs might participate in a wide range of rice growth and development processes beyond biosynthesis of cuticular waxes.

Stagnant deoxygenated growth enhances root suberization and lignifications, but differentially affects water and NaCl permeabilities in rice (Oryza sativa L.) roots

It has been shown that rice roots grown in a stagnant medium develop a tight barrier to radial oxygen loss (ROL), whereas aerated roots do not. This study investigated whether the induction of a barrier to ROL affects water and solute permeabilities. Growth in stagnant medium markedly reduced the root growth rate relative to aerated conditions. Histochemical studies revealed an early deposition of Casparian bands (CBs) and suberin lamellae (SL) in both the endodermis (EN) and exodermis, and accelerated lignification of stagnant roots. The absolute amounts of suberin, lignin and esterified aromatics (coumaric and ferulic acid) in these barriers were significantly higher in stagnant roots. However, correlative permeability studies revealed that early deposition of barriers in stagnant roots failed to reduce hydraulic conductivity (Lp(r) ) below those of aerated roots. In contrast to Lp(r) , the NaCl permeability (P(sr) ) of stagnant roots was markedly lower than that of aerated roots, as indicated by an increased reflection coefficient (σ(sr) ). In stagnant roots, P(sr) decreased by 60%, while σ(sr) increased by 55%. The stagnant medium differentially affected the Lp(r) and P(sr) of roots, which can be explained in terms of the physical properties of the molecules used and the size of the pores in the apoplast.

Mutation in Wilted Dwarf and Lethal 1 (WDL1) causes abnormal cuticle formation and rapid water loss in rice

Epidermal cell layers play important roles in plant defenses against various environmental stresses. Here we report the identification of a cuticle membrane mutant, wilted dwarf and lethal 1 (wdl1), from a rice T-DNA insertional population. The muant is dwarf and die at seedling stage due to increased rates of water loss. Stomatal cells and pavement cells are smaller in the mutant, suggesting that WDL1 affects epidermal cell differentiation. T-DNA was inserted into a gene that encodes a protein belonging to the SGNH subfamily, within the GDSL lipase superfamily. The WDL1–sGFP signal coincided with the RFP signal driven by AtBIP–mRFP, indicating that WDL1 is an ER protein. SEM analyses showed that their leaves have a disorganized crystal wax layer. Cross-sectioning reveals loose packing of the cuticle and irregular thickness of cell wall. Detailed analyses of the epicuticular wax showed no significant changes either in the total amount and amounts of each monomer or in the levels of lipid polymers, including cutin and other covalently bound lipids, attached to the cell wall. We propose that WDL1 is involved in cutin organization, affecting depolymerizable components.