Development and optimization of sequence-tagged microsatellite site markers to detect genetic diversity within Colletotrichum capsici, a causal agent of chilli pepper anthracnose disease

Genomic libraries enriched for microsatellites from Colletotrichum capsici, one of the major causal agents of anthracnose disease in chilli pepper (Capsicum spp.), were developed using a modified hybridization procedure. Twenty-seven robust primer pairs were designed from microsatellite flanking sequences and were characterized using 52 isolates from three countries India, Sri Lanka and Thailand. Highest gene diversity of 0.857 was observed at the CCSSR1 with up to 18 alleles among all the isolates whereas the differentiation ranged from 0.05 to 0.45. The sequence-tagged microsatellite site markers developed in this study will be useful for genetic analyses of C. capsici populations.     

RCN1/OsABCG5, an ATP‐binding cassette (ABC) transporter,is required for hypodermal suberization of roots in rice (Oryza sativa)

Suberin is a complex polymer composed of aliphatic and phenolic compounds. It is a constituent of apoplastic plant interfaces. In many plant species, including rice (Oryza sativa), the hypodermis in the outer part of roots forms a suberized cell wall (the Casparian strip and/or suberin lamellae), which inhibits the flow of water and ions and protects against pathogens. To date, there is no genetic evidence that suberin forms an apoplastic transport barrier in the hypodermis. We discovered that a rice reduced culm number1 (rcn1) mutant could not develop roots longer than 100 mm in waterlogged soil. The mutated gene encoded an ATP‐binding cassette (ABC) transporter named RCN1/OsABCG5. RCN1/OsABCG5 gene expression in the wild type was increased in most hypodermal and some endodermal roots cells under stagnant deoxygenated conditions. A GFP‐RCN1/OsABCG5 fusion protein localized at the plasma membrane of the wild type. Under stagnant deoxygenated conditions, well suberized hypodermis developed in wild types but not in rcn1 mutants. Under stagnant deoxygenated conditions, apoplastic tracers (periodic acid and berberine) were blocked at the hypodermis in the wild type but not in rcn1, indicating that the apoplastic barrier in the mutant was impaired. The amount of the major aliphatic suberin monomers originating from C₂₈ and C₃₀ fatty acids or ω‐OH fatty acids was much lower in rcn1 than in the wild type. These findings suggest that RCN1/OsABCG5 has a role in the suberization of the hypodermis of rice roots, which contributes to formation of the apoplastic barrier.     

ABCG Transporters Are Required for Suberin and Pollen Wall Extracellular Barriers in Arabidopsis

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.     

The role of root apoplastic transport barriers in salt tolerance of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Increasing soil salinity reduces crop yields worldwide, with rice being particularly affected. We have examined the correlation between apoplastic barrier formation in roots, Na⁺ uptake into shoots and plant survival for three rice (Oryza sativa L.) cultivars of varying salt sensitivity: the salt-tolerant Pokkali, moderately tolerant Jaya and sensitive IR20. Rice plants grown hydroponically or in soil for 1 month were subjected to both severe and moderate salinity stress. Apoplastic barriers in roots were visualized using fluorescence microscopy and their chemical composition determined by gas chromatography and mass spectrometry. Na⁺ content was estimated by flame photometry. Suberization of apoplastic barriers in roots of Pokkali was the most extensive of the three cultivars, while Na⁺ accumulation in the shoots was the least. Saline stress induced the strengthening of these barriers in both sensitive and tolerant cultivars, with increase in mRNAs encoding suberin biosynthetic enzymes being detectable within 30 min of stress. Enhanced barriers were detected after several days of moderate stress. Overall, more extensive apoplastic barriers in roots correlated with reduced Na⁺ uptake and enhanced survival when challenged with high salinity. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Water permeability and reflection coefficient of the outer part of young rice roots are differently affected by closure of water channels (aquaporins) or blockage of apoplastic pores

The relative contribution of the apoplastic and cell-to-cell paths to the overall hydraulic conductivity of the outer part of rice roots (LpOPR) was estimated using a pressure perfusion technique for 30-d-old rice plants (lowland cultivar, IR64, and upland cultivar, Azucena). The technique was based on the perfusion of aerenchyma of root segments from two different zones (20-50 mm and 50-100 mm from the root apex) with aerated nutrient solution using precise pump rates. The outer part of roots (OPR) comprised an outermost rhizodermis, an exodermis, sclerenchyma fibre cells, and the innermost unmodified cortical cell layer. No root anatomical differences were observed for the two cultivars used. Development of apoplastic barriers such as Casparian bands and suberin lamellae in the exodermis were highly variable. On average, matured apoplastic barriers were observed at around 50-70 mm from the root apex. Lignification of the exodermis was completed earlier than that of sclerenchyma cells. Radial water flow across the OPR was impeded either by partially blocking off the porous apoplast with China ink particles (diameter 50 nm) or by closing water channels (aquaporins) in cell membranes with 50 micro M HgCl2. The reduction of LpOPR was relatively larger in the presence of an apoplastic blockage with ink ( approximately 30%) than in the presence of the water channel blocker ( approximately 10%) suggesting a relatively larger apoplastic water flow. The reflection coefficient of the OPR (sigmasOPR) for mannitol significantly increased during both treatments. It was larger when pores of the apoplast were closed, but absolute values were low (overall range of sigmasOPR=0.1-0.4), which also suggested a large contribution of the non-selective, apoplastic path to overall water flow. The strongest evidence in favour of a predominantly apoplastic water transport came from the comparison between diffusional (PdOPR, measured with heavy water, HDO) and osmotic water permeability (PfOPR) or hydraulic conductivity (LpOPR). PfOPR was larger by a factor of 600-1400 compared with P(dOPR). The development of OPR along roots resulted in a decrease of PdOPR by a factor of three (segments taken at 20-50 and 50-100 mm from root apex, respectively). Heat-killing of living cells resulted in an increase of PdOPR for both immature (20-50 mm) and mature (50-100 mm) root segments by a factor of two. Even though both pathways (apoplast and cell-to-cell) contributed to the overall water flow, the findings indicate predominantly apoplastic water flow across the OPR, even in the presence of apoplastic barriers. Low diffusional water permeabilities may suggest a low rate of oxygen diffusion across the OPR from aerenchyma to the outer anaerobic soil medium (low PO2OPR). To date, there are no data on PO2OPR. Provisional data of radial oxygen losses (ROL) across the OPR suggest that, unlike water, rice roots efficiently retain oxygen within the aerenchyma. This ability strongly increases as roots/OPR develop.     

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.