Indian Journal of Experimental Biology Vol. 38, October 2000, pp. I 031-1035

Ascorbic acid metabolism in ageing recalcitrant sal (Shorea robusta Gaertri. f.) seeds

K S Krishna Chaitanya, Ranjana Naithani & S C Naithani*

Seed Biology Lab, School of Life Sciences, Pt. Ravishankar Shukla University, Raipur 492 010, India

Received 24 April 1999; revised 23 May 2000

Changes in ascorbate content and its enzymatic utilization pattern were studied in embryonic axes and cotyledons of sal seeds undergoing rapid loss of viability, at ambient conditions. Ascorbate levels were significantly higher initially in the embryonic axes (0.32 mg/g fresh weight) and cotyledons (0.21 mg/g fresh weight) of freshly mature, relatively hydrated (42.2% moi sture content) and I 00% viable sal seeds. It declined sharply as the tissues; embryonic axes and cotyledons, desiccated with absolutely no detectable amount in non-viable seeds (21 % moisture content). Significantly strong correlation was obtained between desiccation of embryonic axes (r = 0.96) and cotyledon (r = 0.97) with loss of ascorbate levels and loss of germinability. Higher rates of ascorbic acid utilization (AAU) recorded in the embryonic axes of I 00% viable seed declined sharply as the seed viability reduced due to desiccation below 36.8 % moisture content. AAU was not detected in the cotyledons.

Desiccation of seeds during storage has frequently been related to oxidative injur/. Free radicals of 0 2

and its derivatives have been suggested to play an important role in declining seed viabilit/'3 mostly by lipid peroxidation4

·5

, protein and nucleic acid damage2

. Plants have developed complex antioxidant system[s] to protect cellular membranes from the damaging effects of "02- radicals6

. Antioxidants like glutathione7

, alpha tocopherol8 and ascorbate9 are considered as chain breaking antioxidants in the free radicals mediated peroxidation due to their free radical scavenging abilities. Ascorbic ac id plays a key role in detoxification of "02- radicals6

. It can react directly by reducing superoxide, hydrogen peroxide, and hydroxyl radical , or quenching singlet oxygen 10

.

Alternatively, it can react indirectly by regenerating alpha tocopherol from alpha chromanoxy radical 10

.

The loss of ascorbate pool resulting from desiccation alone is of pa!"ticular importance and failure to maintain it is an indicator of overall degeneration 'in the capacity to withstand oxidative conditions 11

. Desiccation remarkably reduces ascorbate levels in developing and hydrated seeds12

.

Further, the ascorbate contents, which were similar in desiccation tolerant and sensitive bryophytes reduced as a result of des iccation 11

• Ascorbic acid is known to be acted upon by many enzymes viz., peroxidase 13

,

polyphenol oxidase 14, and specific ascorbic acid

* Correspondent au thor: Email : naithani @mantraonline.com

oxidaset 2. The net results of the achvttles of these

enzymes is termed as ascorbic acid utilization 15•

Higher levels of free ascorbic acid and AAU, leading to higher turnover, have often been correlated with higher growth potential 16 during seed germination.

Our previous work showed that the sal seeds are desiccation-sensitive and deteriorate rapidly due to oxidative stress5

. Massive membrane perturbation caused primarily due to enhanced oxygen free radicals mediated lipid peroxidation has been suggested to be the major event during desiccation induced loss of viability in these seeds5

• 17

•

The present experiments have been carried out on the ascorbic acid content and its utilization in sal seeds undergoing rapid dehydration as a function of ageing, with the intent to examine the role of ascorbic acid and its metabolism during loss of viability.

Materials and Methods Collection of seed - Physiologically mature sal

seeds, 63 days after anthesis5, were collected during

3rd week of May from Gariyabandh Forest (90 km from Raipur) for this study. The Gariyabandh Forest Reserve is situated to the northeast of Raipur and lies between 20°38'N latitude and 82°04' E longitude. Nearly 25-30 elite trees in the forest were marked for collection of fruits and seeds. Seeds were manually plucked and fresh mature seeds (showing I 00% germination within 40-48hr) collected were brought to the laboratory within 4-Shr. The calyces of the

1032 INDIAN J EXP BIOL, OCTOBER 2000

seeds were plucked manually and healthy uninfected seeds of uniform size were sorted out and stored in perforated trays at ambient conditions [40-45% RH and 27-32°C] and used at desired intervals for the analyses .

Moisture content - The moisture content of the seeds , embryon ic axes and cotyledons were estimated as percentage water of fresh weight. Three replicates of 15 seeds each of axes and cotyledons were weighed before and after dryi ng at 80°C for 72hr. The moi sture content was determined following the method given by Internat iona l Seed Testing Association 18

•

Germination assessment - Seeds were surface sterili zed with 0. 1% HgCh solution for 15 mi n and then treated with 0.0 I% dimecron for I Om in to prevent fungal infestat ion. These seeds were then thoroughly washed 5-6 times with glass di sti lled water and then placed in Petri di shes conta in ing distilled water, fo r germination, in dark at 32°-34°C. Germination was scored by emergence of radicle (5-7 mm in length) and expressed as percentage of ,' eeds germinated in each Petri di sh. Five replicates of 10 seeds each were used for germination studies.

Ascorbic acid content and ascorbic acid utilization-The levels of ascorbate and its utilization were measured fo ll owing the method given by Chinoy et al 1

') . The ascorbate was ex tracted by homogeni zi ng 500 mg ti ssue (embryonic axes and cotyledon) in a chilled mortar-pestl e with 2 ml chilled C02 saturated water. The homogenate was transferred to a clean test tube on an ice bath . Two react ion sets were run simultaneously for determining the ascorbate content and its utili zati on. For measuring ascorbate, 1.5 rnl of the homogenate was added to 1.5 ml buffered l-h P03 solution and I ml from thi s mix ture was mixed with 2.5 ml of the dye and the absorbance was recorded at 540 nm. A pinch of ascorbic ac id was added to the mi xture to decolorize the solution and absorbance was reread at 540 nm to determine the turbidity . Ascorbate content was estimated by the diffe rence in the two absorbances.

To determine ascorbic ac id utili zati on (AAU), 1.5 ml of homogenate was added to 1.5 ml ascorbic ac id soluti on and incubated at room temperature in dark for 30 min. To I ml of the incubated mixture, I ml buffered H3PO, was added to stop the reaction. I ml of the reaction mi xture was pipetted out and 2.5 ml dye was added. The absorbance was recorded at 540 nrn. A pinch of ascorbic acid ·was added to the

solution and the absorbance was agam taken at 540nm. The change in absorbance gave the utilizati on of ascorbate added. Ascorbic acid content was ex pressed as mg/g fresh weight sample and ascorbate utilization as mg ascorbic ac id utili zcd/min/g fresh weight.

Results Loss of viability - The freshly mature sal seeds

during storage showed a rapid loss of viability within 6 day of storage at ambient conditi ons (Fig. I). Decline in percentage germination of the seeds was from I 00% initi a ll y to 70% on 4 day and 30% on 5 day to 0% by 6day . A gradual decrease in the percentage moi sture content of the seeds was also di scernible. The moisture content on harves t was 42.2%. The lowest safe moisture content was recorded to be 36.7% on 3day (Fig. I). The moisture content decreased sharply to 2 1% by 6day when no seeds showed germinati on. The fresh seeds showed germination wi thin 40 - 48 hr whereas, showed delayed germination with age ing after 3 days .

The moisture content as well as the rate of des iccation in the spec ific tissues, viz., embryonic axis and cotyledon varied during storage (Fig. 2). T he embryonic axes of I 00% viable seeds recorded hi gher moisture content (54.9 %) as compared to coty ledon (34.4%) from mature freshl y harvested sal seeds. The moisture content of the embryonic axes decli ned sharply to 43.3% on 2 day and finally to 19.8% in the non-viable seeds on 6 day. Desiccat ion of cotyledons exhibited a trend simil ar to the des iccation of embryonic axes. The cotyledons with comparati ve ly lower moi sture registered rap id dec line m moi sture

50 100

~ H+ .r;

"' 40 80 Q) L. ... ~ ~ 0 c ._j 60 0 c

~ Q) 30 c c 0 ·~ u 40 Q) Q) L. (!) .3 20 "' 20 '(5 ~

10 0 0 2 3 4 5 6

Days after harvest

Fig. !- Decline in percen tage germination (• ) and moi sture content (x ) wi th age of mature sal seeds during storage. Each va lue is a mean of 50 observati ons. Vertical bars represent mean ± SD.

CHAIT ANY A et al. : ASCORBIC ACID METABOLISM IN SAL 1033

content from 34.4% on 0 day to 24.7% on 3 day and eventually to as low as 12.2% on 6 day of storage. The rate of desiccation was faster in embryonic axes than the cotyledon.

Ascorbic acid content - The total ascorbate content reduced sharply in both the embryonic axes (Fig. 3) and cotyledons (Fig. 4) with the decline in moisture content during storage. The ascorbic ac id content in the embryonic axes of I 00% viable seeds was hi ghest (0.32 mg/g fresh weight) and decreased gradually with the reducti on in moisture content (Fig. 3). Later, it reduced graduall y to 0. 18 mg/g fresh weight at 36.6% moisture content and the lowest 0 .12 mg/g fresh weight in the embryonic axes of non­viable seeds with 2 1.8% moisture content. No measurable ascorbate content was recorded thereafter. A strong posit ive correlat ion was establi shed between the decli ne in ascorbate levels and loss of viability (r=0.96, P<O.OOl ). Similarly, a dec lining trend in the ascorbate content in the cotyledon of deteriorating sal seeds was discernible, though lower levels of the same were observed in the cotyledonary tissues (Fig. 4) than the embryonic axes . W ith the fa ll in moisture content, there was a rapid loss in the ascorbate content in the cotyledon from 0.2 1 mg/g fresh weight in 100% viable seeds (42.2% moisture content) to 0.1 1 mg/g fresh weight in seeds with 39.3% moisture and no measurable ascorbic ac id was found in the cotyledons with moi sture content as high as 36.7% (3 day). A strong positi ve corre lation was observed between the decl ine in ascorbic acid content and percentage germinati on (r = 0 .97 , P<O.OO I ).

Ascorbic acid utilization - The utilizati on of

60

.... ~ 50

.s::. "' Q) ..... .,_

40 ;g_ 0

~ c 30 ~

c 0 0 Q) 20 ..... :::1 .... "' '(5 10

:.E

0 0 1 2 3 4 5 6

Days after harvest

Fig. 2-Decl ine in moisture content wi th age in embryonic ax is (•) and cotyledon (X) of sal seeds during storage. Vertical bars represent mean ± SD.

ascorbic acid by the seed ti ssues was al so measured (Fig. 5) though no measurable AAU was recorded in the cotyledonary ti ssues. AAU was registered to be 0.53mg ascorbate utilized/min/g fresh weight in the embryonic axes of 0 day seeds wi th 42.2% moisture content. In the initial stages of desiccation , the axia l ti ssues exhibited higher utilization of ascorbate, which later on declined sharply . These levels gradually increased to 0.76 mg ascorbate utili zed/min/g fresh weight with initial desiccation of the seeds to 36.7% moi sture content and thereafter, a steep decline in AAU levels was d iscernible in seeds

0.4 .-------- ----------- ----, 0.4 rAs:..::c.:.:.or::.:b•=te..:.:.m""g/-"-g f::..:re:.:::•hc:....:wt.:..:......._-,

44

Moisture content, % fresh wt

Fig. ]-Relationship between moisture content and ascorbic acid c.:o ntent in the embryonic axis of naturally agei ng sal seeds. Inset : showing the posit ive correlation between loss of viabi lity and ascorbate content in the embryonic ax is (r =0.96, P<O.OO I) . Vert ical bars represent mean ± SD.

0.25 .-----------------------,

0.2

0.15

·:{:::~~ 0.05

0

0.1

0.05 . 100 100 100 100 100 100 90

Germination, •,4 0 L--~-~----_L ____ J_ _ _ _ ~

35 37 39 41 43

Moisture content,% fresh wt

Fig. 4-Decline in ascorbi c acid content in the cotyledon of sal seeds with the relative loss in moisture content. Inset: showing the positive correlation between loss of viability and ascorbate content in the cotyledon (r =0.97, P<O.OOl) . Vertical bars represen t mean ±SD.

1034 INDIAN J EXP BIOL, OCTOBER 2000

.... 0.1 5:

.r: (/) Q) .... 0.08 .... .El c .E c, 0.06 E "0 Q)

~ 0.04 ., ::l Q) .... ra 0.02 .0 .... 0 0 (/)

< 0 19 24 29 34 39 44

Moisture content, %fresh wt

Fig. 5-Relationship between moisture content and ascorbic acid utili zat ion (AAU) in the embryoni c axes of sal seeds. Vert ical

bars represent mean± SD.

with low moisture. AAU was not detected tn non­viable seeds.

Discussion The mature sal seeds ex hibited viability up to 6

days, shortest period so far known , during storage at ambient conditions. Desiccation of seeds below 36.8% leads to rapid loss of viability (Fig. 1). These results confirm our previous findings that the sal seeds being hi ghly desiccation sensitive lose viability very rapidly and are class ified as tme recalcitrant5

.

Significantly higher rates of desiccation were observed in the embryonic axes compared to cotyledon with relatively lower initial moisture content (Fig. 2). The chemical composition of the cotyledon, being rich in oil s, appears to be responsible for lower initial moisture content as also reported in Quercus rubra 20

.

Sharp decline in ascorbate levels recorded in desiccating embryonic axes and cotyledons of sal seeds was similar to the results obtained for other

d 12,21 d 0 0 27 p 0 see s an vegetative tissues -. resence of h1 gher

amounts of ascorbate in freshly harvested sal seeds can be attributed to the unique developmental feature of recalcitrant seeds including seeds of Shorea robusta (Figs 3 and 4) . These seeds do not undergo maturation drying during development and therefore, they are shed with relatively high moisture content23

.

Higher amounts of ascorbate have been shown to be a characteristic feature of developing Vicea faba seeds (with maximum moisture content), and their level s decline concomitantly with the reduction in moisture

d 0 0 J7 content unng maturation -. Further, relatively higher

amounts of ascorbate may effectively suppress the

desiccation induced oxidative stress leading to loss of viability in sal seeds. Remarkably lower levels of ·o2- radical and its mediated lipid peroxidation in 100% viable seeds5 could possibly be due to antioxidant ability of ascorbate to inhibit the lipid peroxidation or directly scavenging · 0 2. radicals. Significantly strong correlation between desiccation of embryonic axes (r = 0.96, P<O.OOI) (Fig. 3- inset) and cotyledons (r = 0.97, P<O.OOl) (Fig. 4 - inset) with loss of ascorbate further establishes the desiccation sensitivity of sal seeds. Comparatively higher amounts of ascorbate along with lower rates of its decline in desiccating embryonic axes than in the cotyledons of ageing sal seeds, appears to be due to differential level of desiccation of these tissues.

Degradation of ascorbate during desiccation has been found to be a common phenomenon in several I

0 72-24 d p ant species- an may be ascribed to a corresponding rise in the ascorbate oxidase activitl 5

.

In sal seeds, the ascorbate utilization is detected only in the embryonic axes and not in the cotyledons. The embryonic axes of I 00% viable seeds showed higher rates of ascorbic acid utilization and as the seed deteriorates (i .e., after 3 days) a sharp loss in ascorbic acid utilization was also discernible, with no ascorbic acid utilization in non-viable seeds (Fig. 5) . The data of Shigeoka et al?6

, Tommasi et al. 27 and Arrigoni et al. 12 suggested that decrease in ascorbate and ascorbate utilization occurring during ageing can be correlated with the onset of biochemical pathway leading to loss of viabi lity in seed 28

.

Acknowledgment The authors acknow ledge Prof. M L Naik, Head,

School of Life Sciences, Pt. Ravi shankar Shukla University, Raipur for providing necessary laboratory facilities. Financial assistance to KSK by Madhya Pradesh Council of Science & Technology, Bhopal (B-47/90) is al so acknowledged.

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CHAIT ANY A et al.: ASCORBIC ACID METABOLISM IN SAL 1035

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