Yield and Quality of Green Leafy Vegetables in Postharvest

CHAPTER II 
Exogenous Proline Improves Quality Postharvest of rocket leaves upon 
salinization 
 
1. Introduction 
Rocket (Diplotaxis tenuifolia (L.) DC.) is a member of the family Brassicaceae found in 
most Mediterranean countries. Rocket is a cruciferous crop used extensively in baby leaf 
salads. Like other cruciferous vegetables, rocket contains a range of health-promoting 
phytonutrients including vitamin C, fiber, flavonoids, carotenoids and glucosinolates 
(Barillari et al., 2005; Crozier et al., 1997; Kris-Etherton et al., 2002; Van Poppel et al., 
1999). Many phytochemicals, including glucosinolates, phenols, and flavonoids are 
pungent and bitter tasting, which poses a quandary for a food industry intent on raising the 
levels of these compounds in their products to boost perceived health attributes 
(Drewnowski and Gomez-Carneros, 2000). 
The discovery of bioactive components in foods is exciting, suggesting the possibility of 
improved public health through diet. Yet the content of bioactive components in plant food 
varies, making quality control and intake recommendations problematic. Variation in 
content of bioactive components in fruits and vegetables depends upon both genetics and 
environment, including growing conditions, harvest and storage. Cruciferous vegetables, 
which contain both anticarcinogenic and antioxidant properties, are excellent examples to 
illustrate the problem in assessing health benefits of foods that vary in content of bioactive 
components (Jeffrey et al., 2003). 
Some nutrients, such as the antioxidant vitamins, carotenoids, tocopherols and ascorbic 
acid, appear to play a dual role in metabolism. At one dose they are required for normal 
growth and development, and at a higher dose they appear to provide antioxidant 
 16
protection against chronic diseases, including chronic heart disease, arthritis and cancer 
(Krinsky et al., 2000). 
Green leafy vegetables have significantly higher total carotene content and highest total 
phenols than other vegetables. The major carotenoids in green leafy vegetables are lutein 
and all-trans-β-carotene (Cho et al., 2007). 
Vitamin C is a universal constituent of all green leaf plants, its minimum intake must be of 
60mg day for adults. In crop plants, vitamin C content is influenced by seasonal factors 
such as sowing time and harvesting date and is often used as a ‘marker’ for postharvest 
deterioration (Sood Mand Malhotra 2001). It develops an important role in photosynthesis 
and photoprotection, in defence against ozone and other oxidative stresses (Smirnoff 
1996). 
Ascorbate occurs in the cytosol, chloroplasts, vacuoles, mitochondria and cell wall 
(Anderson, et al., 1983; Rautenkranz et al., 1994). The concentration in chloroplasts can be 
high and is probably related to its central role in photosynthesis (Foyer, 1993). 
Consumption of lightly processed vegetables was increased over the last decade. More 
research was done to identify better cultivation and storage conditions that will minimize 
nutrient and quality losses in these highly perishable products. 
Fresh-cut (minimally-processed) horticultural products are subjected to simple operations 
soon after harvest, such as cleaning, washing, cutting and packaging, which make them 
ready-to-use. It is well known that from the time of harvest quality declines in fruit and 
vegetables and many nutrients are lost rapidly. This quality decline includes visual 
symptoms, such as loss of turgor and yellowing of green produce, as well as loss of 
important nutrients, such as sugars and vitamin C (Gil et al., 1999; Lee and Kader, 2000). 
Physical damage during preparation causes an increase in respiration rates, biochemical 
changes and microbial spoilage, which may result in degradation of colour, texture and 
flavour of the produce (Cantwell, 1996). In other reports, minimal processing increases 
 17
phenolic metabolism and therefore, the accumulation of phenolic metabolites (Tomas-
Barberan et al., 2000). Moreover, the content of phenols is associated with the sensitivity 
to enzymatic browning in several vegetables (Brecht et al., 2004). 
It is well known that different stresses, location climates, microenvironments and physical 
and chemical stimulus (often called elicitors) qualitatively and quantitatively alter the 
content of bioactive secondary metabolites, and whole-plant elicitation increases the 
amounts of bioactive compounds in foods of plant origin. (Demmig-Adams and Adams 
2002; Van Dam et al., 2004). 
A elicitor could to be considered the proline, which it accumulation in the cell (up to 80% 
of the amino acid pool under stress and 5% under normal conditions) due to increased 
synthesis and decreased  degradation under a variety of stress conditions such as salt and 
drought (Delauney and Verma, 1993; Bohnert and Jensen, 1996; Kavi Kishor, 1988; Kohl 
et al., 1991; Parida and Das, 2005; Sarvesh et al., 1996; Schat et al., 1997; Trotel et al., 
1996). 
Tolerance to abiotic stress, especially to salt stress, was observed in a variety of transgenics 
that were engineered for overproduction of proline (Kavi Kishor et al., 1995; Roosens et 
al., 2002). Proline seems to have diverse roles under osmotic stress conditions, such as 
stabilization of proteins, membranes and subcellular structures (Vanrensburg et al., 1993), 
and protecting cellular functions by scavenging reactive oxygen species (Bohnert and 
Shen, 1999; Samaras et al., 1995; Tsugane et al., 1999; Hong et al., 2000; Okuma et al., 
2000, 2002; Heuer, 1994). Among various compatible solutes, proline is the only molecule 
that has been shown to protect plants against singlet oxygen and free radical induced 
damages (Alia et al., 1997). Since proline can act as a singlet oxygen quencher (Alia and 
Pardha Saradhi, 1993), and as a scavenger of OH· radicals, it is able to stabilize proteins, 
DNA as well as membranes (Matysik et al., 2002).  
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Salt stress causes significant crop losses, it is the major environmental factor limiting plant 
growth and productivity (Allakhverdiev et al., 2000), through lowering of the 
photosynthesis and the production of actived oxygen radicals such hydrogen peroxide, 
superoxide, singlet oxygen and hydroxyl (Smirnoff, 1995).  
The nitrate levels in leafy vegetables can be of considerable importance for their quality 
owing to its potential harmful effects on human health by leading to methaemoglobinaemia 
or forming nitrosamines and nitrosamides by reaction with amines and amides after nitrate 
reduction (Marschner, 1997; Walker, 1990; Bianco et al., 1998; Santamaria et al., 2002). 
The acceptable daily intake of nitrate was set at 0–3.65 mg/kg body weight by the 
European Communities’ Scientific Committee for Food in 1992. One of the problems in 
rocket commercialization is the high nitrate content of fresh product, critical for human 
health. Besides, alternative cultural techniques are required to control mineral nutrition, 
with the aim of standardizing the qualitative characteristics of Leafy Vegetable Production 
(Incrocci et al., 2001). 
The objective of the work was to set up a Soilless Culture System to grow rocket 
comparing different growing practices to obtain a fresh-cut product of high quality in terms 
of marketable standards and shelf life. 
 
2. Materials and methods 
The experiment was carried out at the University of Naples Federico II (40°49’ N, 14° 15’ 
E, 30 m.a.s.l.) in a cold glasshouse and were implemented two cycle of seeding (spring and 
summer).  
The first was from 18 March to 24 April 2008 (seed-to-harvest time was 39 days), while 
the second from 5 June to 2 July 2008 (seed-to-harvest time was 27 days). The rocket was 
sown in 4 m2 polystyrene containers filled with sterilised soil and grown in floating 
systems. Each growing unit was filled with 1.000 L of aerated nutrient solution. 
 19
Oxygenation of the solution was maintained with electric pumps. Planting density was 100 
plants m
-2
, as commercially adopted for hydroponic production of rocket. The composition 
of the standard nutrient solution adopted in all experiments was N-NO
- +
 14 mM; N-NH
3 4
 6 
mM; Cl
- -
 3.0 mM; PO
4
 3.5 mM; S 6.0 mM; Ca 5.0 mM; Mg 3.7 mM; K 10.5 mM; Na 2.2 
mM; Bo: 0.02 mM; Fe 0.04 mM (Pimpini et al., 2001; Marschner, 1995). Starting from 26 
DAS (first cycle) and from 19 DAS (second cycle), the leaves of plants were sprayed with 
solution of proline 20 mM in distilled water and distilled water (control). After 24 hours, 
the plants were exposed to a salt stress of 0 (control), 25, 50 and 100 mM NaCl. Two days 
before harvest, the second leafy treatment was applied at plants. The experimental design 
was a randomized block with two replications.  
Determinations and analysis: Plants were harvested on April 24 and on July 2. At harvest, 
the fresh products were manually cleaned, by discarding stems, damaged leaves and yellow 
inflorescences. Intact leaves of the first harvest were accurately washed in order of three 
times with microbiologically pure water (spring crop), while in the second harvest, the 
leaves were washed with ascorbic acid (150 mg l
-1
) (summer crop) and both air flow dried 
on a strainer. The leaves were subsequently kept in plastic boxes. The containers were 
wrapped with a multistrate film (PET 12+ COEX/EVOH/PE 95), highly impermeable to 
gases and water vapour, with permeability to O2 < 5 dm3 m-2 d-1 bar-1at 23 °C and 0% 
U.R. and to H2O < 5g m-2 d-1 bar-1 at 38 °C and 100% U.R. (Gruppo Fabbri S.P.A., 
Modena, Italy).  
The containers were kept at 4 °C for 6 days, under two storage regimes: 1) dark conditions 
(control); 2) daily 8-hour exposure to a relatively low light intensity(PPFD=16 µmol m
-2 
-1
s ). 
Leaf samples were collected on day 0, 2, 4 and 6. During storage, loss weight of the 
packaging was also evaluated. Leaf dry weights upon oven dehydration at 60°C (until 
steady weight) and dry matter were measured.  
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