Water Relations of Seed Development and Germination in Muskmelon (Cucumis melo L.) : I. Water Relations of Seed and Fruit Development

Total water potential (ψ), solute potential, and turgor potential of field-grown muskmelon (Cucumis melo L.) fruit tissue (pericarp) and seeds were determined by thermocouple psychrometry at 5-day intervals from 10 to 65 days after anthesis (DAA). Fruit maturity occurred between 44 and 49 DAA, and seed germination ability developed between 35 and 45 DAA. Pericarp ψ was essentially constant at approximately −0.75 megapascal (MPa) from 10 to 25 DAA, then decreased to a minimum value of −1.89 MPa at 50 DAA before increasing to −1.58 MPa at 65 DAA. Seed ψ remained relatively constant at approximately −0.5 MPa from 10 to 30 DAA then decreased to −2.26 MPa at 50 to 60 DAA before increasing to −2.01 MPa at 65 DAA. After a rapid increase to 20 DAA, seed fresh weight declined until 30 DAA due to net water loss, despite continuing dry weight gain. As fruit and seed growth rates decreased, turgor potential initially increased, then declined to small values when growth ceased. A disequilibrium in ψ was measured between seeds and pericarp both early and late in development. From 20 to 40 DAA, the ψ gradient was from the seed to the tissue, coinciding with water loss from the seeds. From 50 to 65 DAA, seed ψ decreased, causing a reversal of the ψ gradient and a slight increase in seed water content. The partitioning of solutes between symplast and apoplast may create and maintain ψ gradients between the pericarp and seed. The low solute potential within the pericarp due to solute accumulation and loss of cellular compartmentation during ripening and sensecence may be involved in prevention of precocious germination of mature seeds.     

Developmental control of xylem hydraulic resistances and vulnerability to embolism in Fraxinus excelsior L.: impacts on water relations

The freezing tolerance of many plants, such as pea (Pisum sativum),is increased by exposure to low temperature or abscisic acidtreatment, although the physiological basis of this phenomenonis poorly understood. The freezing tolerance of pea shoot tips,root tips, and epicotyl tissue was tested after cold acclimationat 2C, dehydration/rehydration, applications of 10^–4M abscisic acid (ABA), and deacclimation at 25C. Tests wereconducted using the cultivar ‘Alaska’, an ABA-deficientmutant ‘wil’, and its ‘wildtype’. Freezinginjury was determined graphically as the temperature that caused50% injury (T₅₀) from electrical conductivity. Endogenous ABAwas measured using an indirect enzyme-linked immunosorbant assay,and novel proteins were detected using 2-dimensional polyacrylamidegel electrophoresis. The maximum decrease in T₅₀ for root tissuewas 1C for all genotypes, regardless of treatment. For ‘Alaska’shoot tips and epicotyl tissue, exogenous ABA increased thefreezing tolerance by –1.5 to –4.0C, while coldtreatment increased the freezing tolerance by –7.5 to–14.8C. Cold treatment increased the freezing toleranceof shoot tips by –9 and –15C for ‘wil’and ‘wild-type’, respectively. Cold acclimationincreased endogenous ABA concentrations in ‘Alaska’shoot tips and epicotyls 3- to 4-fold. Immunogold labeling increasednoticeably in the nucleus and cytoplasm of the epicotyl after7 d at 2C and was greatest after 30 d at the time of maximumfreezing tolerance and soluble ABA concentration. Cold treatmentinduced the production of seven, three, and two proteins inshoot, epicotyl, and root tissue of ‘Alaska’, respectively.In ‘Alaska’ shoot tissue, five out of seven novelproteins accumulated in response to both ABA and cold treatment.However, only a 24 kDa protein was produced in ‘wil’and ‘wild-type’ shoot and epicotyl tissues aftercold treatment. Abscisic acid and cold treatment additivelyincreased the freezing tolerance of pea epicotyl and shoot tissuesthrough apparently independent mechanisms that both resultedin the production of a 24 kDa protein. Key words: Pisum sativum, cold acclimation, immuno-localization     

Weakening of muskmelon perisperm envelope tissue 4

Muskmelon (Cucumis melo L.) embryos are enclosed in an envelopeof tissue consisting of a layer of endosperm and a multi-cell-layeredperisperm that the radicle must penetrate for germination tooccur. The force and energy required to penetrate the perispermenvelope tissue were measured using an Instron universal testingmachine at a crosshead speed of 5 mm min^–1 after 0, 10,15, 22, 23, and 25 h of imbibition at 25C. The cellular structureof perisperm envelope tissue surrounding the radicle was observedafter 10, 15, 20, 25, and 48 h of imbibition using scanningelectron microscopy. The force required to puncture 5-mm-long,micropylar seed pieces declined steadily from 1.65 N in driedseeds to 0.65 N after 21 h of imbibition. The penetration energydeclined from 3.0 N mm in dry seeds to 1.1 N mm at 21 h afterthe start of imbibition when the first seeds germinated. Theforce and energy required to penetrate germinated seed pieceswere 0.55 N and 0.9 N mm, respectively, so the net punctureforce and energy needed to rupture the micropylar region ofthe perisperm envelope was roughly 0.10 N and 0.2 N mm at radicleemergence, respectively. Instron measurements of penetrationforce and energy decreased dramatically at crosshead speedsless than the 5 mm min^–1. Crosshead speeds greater than5 mm min^–1 may overestimate the pressure needed to ruptureperisperm and endosperm tissues. Intracellular cracks were firstobserved in SEM images 15 h after the start of imbibition, andafter 20 h cracking was apparent throughout the micropylar regionof the perisperm envelope. The perisperm envelope ruptured inone of two ways, coincident with radicle emergence. In approximately85% of muskmelon seeds, a large crack formed in the perispermenvelope adjacent to the radicle, while in roughly 15 % a circulararea of the perisperm envelope detached during radicle emergence.In dead seeds, the penetration force remained constant from10–24 h after the start of imbibition, and there wereno visible signs of tissue degradation. Cellular degradationand weakening of the perisperm envelope tissue precedes radicleemergence in muskmelon seeds. Key words: Seed, Instron, turgor, cell wall, electron microscopy, Cucumis melo     

Nicotiana species as surrogate host for studying the pathogenicity of Acidovorax citrulli,the causal agent of bacterial fruit blotch of cucurbits

Bacterial fruit blotch (BFB) caused by Acidovorax citrulli is one of the most important bacterial diseases of cucurbits worldwide. However, the mechanisms associated with A. citrulli pathogenicity and genetics of host resistance have not been extensively investigated. We idenitfied Nicotiana benthamiana and Nicotiana tabacum as surrogate hosts for studying A. citrulli pathogenicity and non-host resistance triggered by type III secreted (T3S) effectors. Two A. citrulli strains, M6 and AAC00-1, that represent the two major groups amongst A. citrulli populations, induced disease symptoms on N. benthamiana, but triggered a hypersensitive response (HR) on N. tabacum plants. Transient expression of 19 T3S effectors from A. citrulli in N. benthamiana leaves revealed that three effectors, Aave_1548, Aave_2708, and Aave_2166, trigger water-soaking-like cell death in N. benthamiana. Aave_1548 knockout mutants of M6 and AAC00-1 displayed reduced virulence on N. benthamiana and melon (Cucumis melo L.). Transient expression of Aave_1548 and Aave_2166 effectors triggered a non-host HR in N. tabacum, which was dependent on the functionality of the immune signalling component, NtSGT1. Hence, employing Nicotiana species as surrogate hosts for studying A. citrulli pathogenicity may help characterize the function of A. citrulli T3S effectors and facilitate the development of new strategies for BFB management.     

Water Relations of Seed Development and Germination in Muskmelon (Cucumis melo L.) : III. Sensitivity of Germination to Water Potential and Abscisic Acid during Development

Muskmelon (Cucumis melo L.) seeds are germinable 15 to 20 days before fruit maturity and are held at relatively high water content within the fruit, yet little precocious germination is observed. To investigate two possible factors preventing precocious germination, the inhibitory effects of abscisic acid and osmoticum on muskmelon seed germination were determined throughout development. Seeds were harvested at 5-day intervals from 30 to 65 days after anthesis (DAA) and incubated either fresh or after drying on factorial combinations of 0, 1, 3.3, 10, or 33 micromolar abscisic acid (ABA) and 0, −0.2, −0.4, −0.6, or −0.8 megapascals polyethylene glycol 8000 solutions at 30°C. Radicle emergence was scored at 12-hour intervals for 10 days. In the absence of ABA, the water potential (Ψ) required to inhibit fresh seed germination by 50% decreased from −0.3 to −0.8 megapascals between 30 and 60 DAA. The Ψ inside developing fruits was from 0.4 to 1.4 megapascals lower than that required for germination at all stages of development, indicating that the fruit Ψ is sufficiently low to prevent precocious germination. At 0 megapascal, the ABA concentration required to inhibit germination by 50% was approximately 10 micromolar up to 50 DAA and increased to >33 micromolar thereafter. Dehydration improved subsequent germination of immature seeds in ABA or low Ψ. There was a linear additive interaction between ABA and Ψ such that 10 micromolar ABA or −0.5 megapascal osmotic potential resulted in equivalent, and additive, reductions in germination rate and percentage of mature seeds. Abscisic acid had no effect on embryo solute potential or water content, but increased the apparent minimum turgor required for germination. ABA and osmoticum appear to influence germination rates and percentages by reducing the embryo growth potential (turgor in excess of a minimum threshold turgor) but via different mechanisms. Abscisic acid apparently increases the minimum turgor threshold, while low Ψ reduces turgor by reducing seed water content.     

Water Relations of Seed Development and Germination in Muskmelon (Cucumis melo L.) : V. Water Relations of Imbibition and Germination

The initiation of radicle growth during seed germination may be driven by solute accumulation and increased turgor pressure, by cell wall relaxation, or by weakening of tissues surrounding the embryo. To investigate these possibilities, imbibition kinetics, water contents, and water (Ψ) and solute (ψ_s) potentials of intact muskmelon (Cucumis melo L.) seeds, decoated seeds (testa removed, but a thin perisperm/endosperm envelope remains around the embryo), and isolated cotyledons and embryonic axes were measured. Cotyledons and embryonic axes excised and imbibed as isolated tissues attained water contents 25 and 50% greater, respectively, than the same tissues hydrated within intact seeds. The effect of the testa and perisperm on embryo water content was due to mechanical restriction of embryo swelling and not to impermeability to water. The Ψ and ψ_s of embryo tissues were measured by psychrometry after excision from imbibed intact seeds. For intact or decoated seeds and excised cotyledons, Ψ values were >−0.2 MPa just prior to radicle emergence. The Ψ of excised embryonic axes, however, averaged only −0.6 MPa over the same period. The embryonic axis apparently is mechanically constrained within the testa/perisperm, increasing its total pressure potential until axis Ψ is in equilibrium with cotyledon Ψ, but reducing its water content and resulting in a low Ψ when the constraint is removed. There was no evidence of decreasing ψ_s or increasing turgor pressure (Ψ-ψ_s) prior to radicle growth for either intact seeds or excised tissues. Given the low relative water content of the axes within intact seeds, cell wall relaxation would be ineffective in creating a Ψ gradient for water uptake. Rather, axis growth may be initiated by weakening of the perisperm, thus releasing the external pressure and creating a Ψ gradient for water uptake into the axis. The perisperm envelope contains a cap of small, thin-walled endosperm cells adjacent to the radicle tip. We hypothesize that weakening or separation of cells in this region could initiate radicle expansion.     

Compartmentation of solutes and water in developing sugarcane stalk tissue

Previous studies have suggested that the apoplast solution of sugarcane stalk tissue contains high concentrations of sucrose, but the accuracy of these reports has been questioned because sucrose leakage from damaged cells may have influenced the results. In this study, the solute potential of the apoplast and symplast of the second (immature), tenth, twentieth, thirtieth, and fortieth internodes of field-grown sugarcane (Saccharum spp. hybrid) stalk tissue was determined by two independent methods. Solute potential of the apoplast was measured either directly by osmometry from solution collected by centrifugation, or inferred from the initial water potential of fully hydrated tissue determined by thermocouple psychrometry before the tissue was progressively dehydrated for generation of water potential isotherms. Both methods produced nearly identical values ranging from −0.6 to −1.8 megapascals for immature and mature tissue, respectively. The solute potential of the symplast determined by either method ranged from −1.0 to approximately −2.2 megapascals for immature and mature internodes, respectively. Solute quantitation by HPLC agreed with concentrations inferred from osmometry. Washing thirtieth internode tissue in deionized water increased pressure potential from 0.29 to 1.96 megapascals. The apoplast of mature sugarcane stalk tissue is a significant storage compartment for sucrose containing as much as 25% of the total tissue water volume and as much as 21% of the stored sucrose.