J. AMER. Soc. HORT. SCI. 116(6):1019-1024. 1991. Carbohydrate Depletion and Leaf Blackening in . Protea neriljiolia Robyn McConchiel and N. Suzanne Lang2 Department of Horticulture, Julian C. Miller Hall, Louisiana State University, Baton Rouge, LA 70803 Ken C. Gross . U. S. Department of Agriculture, Agriculture Research Service, Horticultural Crops Quality Laboratory, Beltsville Agricultural Research Center-West, Beltsville, MD 20705 Additional index words. cut flowers, carbon partitioning, photosynthesis, source-sink Abstract. Leaf blackening on cut flower Protea nerii[olia R. Br. stems was dramatically reduced under a 12-hour photosynthetic light period (120 µmol·m-2·s-1) at 25C for 15 days compared with stems kept in the dark. In the light, addition of 0.5% exogenous sugar to the vase solution resulted in a maximum of 2.5% leaf blackening, while stems with no exogenous sugar had a maximum of 16.5%. Continuous darkness resulted in 94% leaf blackening by day 7, irrespective of sugar treatment. Starch and sucrose concentrations were markedly lower in leaves on dark- held stems than in leaves on stems held in the light; thus, carbohydrate depletion could be the primary stress that initiates leaf blackening. In the light, rates of carbon exchange and assimilate export were similar, indicating that the amount of carbon fixed maybe regulated by sink demand. The pattern of carbon partitioning changed in light- held leaves of the 0% sugar treatment during rapid floral expansion and senescence. Inflorescence expansion appears to influence partitioning of photoassimilates and storage reserves into transport carbohydrates; under decreased sink demand, the assimilate export rate decreases and photoassimilates are partitioned into starch. The data suggest that sink strength of inflorescences held in darkness may be responsible for the depletion of leaf carbohydrates and. consequently, blackening. Many Proteaceous species have become important “cut-flower The in florescence of Protea spp. is classified as an involu- crops. A major postharvest problem of this genus, in particular crate capitulum, consisting of multiple individual flowers aris- Protea neriifolia and P. eximia, is premature blackening of the ing from inside the involucral bracts (Johnson and Briggs, 1975). leaves (Brink and de Swardt, 1986; Ferreira, 1983; Newman et To maximize inflorescence vase life, the Protea inflorescence al., 1989; Paull et al., 1980). Leaves of susceptible species is generally harvested when the involucral bracts are beginning begin to turn black within 3 to 7 days after harvest, severely to unfold. At this stage, many of the flowers inside the bracts reducing the market value of a flower stem that otherwise has are immature and have a high respiratory requirement to com- a potential vase life of 3 to 4 weeks. Leaf blackening appears plete development (Ferreira, 1986) and produce nectar. Thus, to be caused by oxidation of the polyphenols and leuco-antho- the developing in florescence is probably a very strong sink that cyanins (Whitehead and de Swardt, 1982) that leak into the cannot be supported adequately by carbohydrate pools in source cyfosol from the vacuole after rupture of intracellular mem- leaves and stems of cut flowers under dark postharvest condi- branes (Brink and de Swardt, 1986; Ferreira, 1986; Newman tions. et al., 1989). The physiological mechanisms or stresses that Despite the evidence that depleted carbohydrate reserves may initiate rupture of the intracellular membrane and subsequent result in the onset of leaf blackening, carbohydrate metabolism leaf blackening have not been clearly established. and partitioning have not been examined during a typical post- However, leaf blackening is known to decrease significantly harvest period. Investigation of photosynthetic rates and parti- when the influence of the inflorescence sink is eliminated by tioning of soluble and insoluble nonstructural carbohydrates in girdling the stem immediately below the inflorescence or by response to altered floral sink demand may help clarify some removing it (Newman et al., 1989; Paull et al., 1980). Black- of the physiology of leaf blackening. Here we report on the ening is also reduced by lighted postharvest conditions (New- carbohydrate metabolism of P. neriifolia stems used as cut flow- man et al., 1989) and the addition of external sugar to the vase ers under light and dark conditions to determine whether de- solution at concentrations ranging from 0.5% to 1.0% (Brink pleted leaf reserves are related to the onset of leaf blackening. and de Swardt, 1986; Ferreira, 1986; Newman et al., 1989). In addition, we investigated the effect of an external carbohy- These observations suggest that depletion of carbohydrates from drate source (sucrose) on carbohydrate metabolism and leaf leaves, caused by translocation to the strong inflorescence sink, blackening. may play a role in initiating the cascade of events that leads to leaf blackening. Materials and Methods Uncut vegetative plant material. To determine the typical nonstructural carbohydrate profile of intact plants, samples were Received for publication 25 Mar. 1991. Paper no. 91-28-5169 of the Journal taken from plants of ‘Pink Ice’ P. nenifolia grown in a green- Series, Louisiana Agricultural Experiment Station and Louisiana State Univ. house ( »25C day/12C night) in 19-liter containers filled with Agriculture Center. We are grateful to V. Lancaster, Kansas State Univ., for statistical assistance and J. Nelson, Goleta, Calif., for donation of flowers. The 1 scoria : 1 peat : 1 perlite (by volume). Plants were watered cost of publishing this paper was defrayed in part by the payment of page weekly and fertilized with blood meal (12N–OP–OK) and am- charges. Under postal regulations, this paper therefore must be hereby marked monium sulfate (21 N–OP–OK) (Hi-Yield Fertilizers, Volunta~ advertisement solely to indicate this fact. lGraduate Research Assistant. ‘Assistant Professor. Abbreviations: AER, assimilation export rate; R, carbon exchange rate. J. Amer. Soc Hort. Sci. 116(6):1019-1024. 1991. 1019 Purchasing Groups, Bonham, Texas). Based on uniformity, one 80% ethanol (v/v) and evaporated to dryness according to the vegetative stem was selected on each of four plants and divided method of Robbins and Pharr (1988). The samples were redis- into 10 phyllotactic divisions (one division equals one complete solved in 2 ml of 80’% ethanol, and soluble carbohydrates per spiral of leaves) beginning at the distal end. Four leaf punches unit leaf area were determined using high-performance liquid (0.5 cm in diameter) were taken from four randomly selected chromatography (HPLC) as described by Picha (1985). Sugars leaves in each phyllotactic division on the stems and analyzed were identified and quantified based on retention times and areas for soluble and insoluble nonstructural carbohydrates. under the peak for the following sugar standards: 0.4% xylose, Postharvest cut flower stems. Flowering P. neriifolia stems 0.1% fructose, O. 1% glucose, 0.2% sucrose, and 0.1% maltose. were harvested in Goleta, Calif., during late Sept. 1989 and For further analysis of soluble carbohydrates, the dried sam- air-freighted to Baton Rouge, La. Stems were then recut (1 cm), ples of HPLC fractions were made into their alditol acetate placed in 1 liter of deionized distilled water containing 50 ppm derivatives as described by Blakeney et al. (1983). Monosac- hypochlorite plus 0.0% or 0.5% sucrose (hereafter referred to charide derivatives were then separated on a 25-m 5% phenyl- as O% and 0.5% sugar treatments), and the number of leaves methylsilicone column (0.2 mm id. ) using capillary gas was recorded. Vase solutions were changed every 2 days to chromatography conditions as described by Gross (1986). coincide with sampling. Monosaccharide derivatives were detected by gas chromatog- Leaf blackening was determined at each sampling by counting raphy/mass spectrophotometry (GC/MS) and the spectra com- the number of leaves with 10% or more of the leaf area black- pared with standards. ened. Inflorescence diameter was measured to determine the The pellet from the soluble carbohydrate ethanolic extaction progress of inflorescence development. The onset of senescence was used for starch analysis. Starch concentrations were deter- was determined visually, based on browning of the involucral mined enzymatically by detecting released glucose (Robbins and bracts. Pharr, 1988). Stems were analyzed under light and dark postharvest con- Statistical analysis. Each data set was analyzed as a repeated ditions for carbon exchange rates (CER), assimilate export rate measures design with a one-way treatment structure (0.5% sugar (AER), and leaf carbohydrate composition. The limited number or O% sugar) (MilIiken and Johnson, 1984). Measurements were of leaves within a phyllotactic division, as well as on the whole taken every 2 days for 7 days in the dark experiment (by which stem of the commercially harvested stems, precluded us from time all leaves had blackened) and 15 days for the light exper- sampling on a phyllotactic basis over the 15-day postharvest iment. Light and dark experiments could not be compared di- period. rectly since replicate environmental growth chambers were Dark and light experiments. Four stems’ randomly selected unavailable. from each sugar treatment were placed in a growth chamber (Environmental Growth Chambers, Chagrin Falls, Ohio) at 25 Results ± IC in either continuous darkness or a photosynthetically ac- tive radiation (PAR) of 120 µmols·m-2·.s-l for 12 h each There was no wounding response detected on treatment stems 24-h interval. To ensure that removal of leaf disk samples did compared to the nonwounded controls in either the light or dark not confound other measurements, a nonwounded control was experiments (data not shown). included for each sugar treatment. This control consisted of a Dark experiment. The onset of leaf blackening under contin- complete replication of stems for which all developmental and uous darkness at 25C was rapid (Fig. 1). By day 7, > 94% of photosynthetic characteristics were measured, but no leaf disks the leaves in both treatments had at least 10% leaf area black- were removed.