Shrunken-i Encoded Sucrose Synthase Is Not Required for ... - NCBI

Plant Physiol. (1988) 88, 1219-1221 0032-0889/88/88/1219/03/$0 1.00/0

Shrunken-i Encoded Sucrose Synthase Is Not Required for Sucrose Synthesis in the Maize Endosperm1 Received for publication March 25, 1988 and in revised form June 28, 1988

B. GREG COBB2* AND L. CURTIS HANNAH Department of Vegetable Crops, Institute of Food and Agricultural Sciences, University of Florida Gainesville, Florida 32611 Since this molecule is not hydrolyzed by invertase this suggests that hydrolysis of sucrose is not a requirement for uptake. Kernels of wild-type maize (Zea mays L.) shrunken-i (shi), deficient Perhaps unexpectedly, maize kernels have the ability to grow in the predominant form of endosperm sucrose synthase and shrunken-2 and develop into viable seeds when sucrose is replaced with (sh2), deficient in 95% of the endosperm ADP-glucose pyrophosphoryl- reducing sugars (3). This observation clearly shows that sucrose ase were grown in culture on sucrose, glucose, or fructose as the carbon is not essential for kernel development and raises the possibility source. Analysis of the endosperm extracts by gas-liquid chromatography that there is some interconversion of the various sugars that revealed that sucrose was present in the endosperms of all genotypes, come early in the metabolic path. Consistent with this are the regardless of carbon supply, indicating that all three genotypes are earlier observations of Shannon and coworkers (4, 13-15) who capable of synthesizing sucrose from reducing sugars. The finding that showed that sucrose can be broken down upon entry into the sucrose was present in shl kernels grown on reducing sugars is evidence maize seed. Their data further suggest that sucrose may be that shrunken-i encoded sucrose synthase is not necessary for sucrose resynthesized in the endosperm cells before entry into the starch synthesis. Shrunken-i kernels developed to maturity and produced viable biosynthetic pathway. seeds on all carbon sources, but unlike wild-type and sh2 kernels grown By use of the in vitro kernel development scheme of Gengenin vitro, sucrose was not the superior carbon source. This latter result bach (6) as modified by us (2) we can ask very simple and directly provides further evidence that the role of sucrose synthase in maize whether kernels have the ability to synthesize sucrose when they endosperm is primarily that of sucrose degradation. are allowed to develop on reducing sugars. This is the subject of the following report. The data clearly show that kernels of the wild-type genotype, as well as shI and sh2, can synthesize sucrose from reducing sugars. Since this conversion is not reduced in the shI mutant, deficient in the predominant SS3 (UDPG: 1-fructose 2-glucosyl-transferase, EC 2.4.1.13), this enzyme must not be In cereals, the metabolic machinery involved in utilizing the necessary for the observed conversion. Indeed, data presented hexoses of sucrose which enters the base of the seed and ulti- here support the view that the physiological role of SS is predommately converts that carbon into starch in the endosperm has inantly that of sucrose degradation. been the subject of many investigations. In attempts to identify and order the intermediates involved in this long and compliMATERIALS AND METHODS cated pathway, studies have recently focused on sucrose and the Plant Material. Ears of shl, sh2, and wild-type maize (Zea number of times it appears in the flow of carbon through this path. While it is clear that sucrose is the form of sugar that enters mays L.) were hand pollinated and harvested at 5 dpp. These the seed, it still remains an open question whether sucrose is were transported on ice and held at 4°C until processed. The resynthesized from C-6 sugars in the process of starch synthesis. outer husks were removed, the ear sprayed with ethanol and Jenner (8, 9) using asymmetrically labeled sucrose, concluded flamed. Ear sections, consisting of 2 rows of 8 kernels, were that sucrose was not broken down and resynthesized in the removed and placed in media containing sucrose (150 g L'), developing wheat kernel. In the case of maize endosperm we glucose (150 g L`), or fructose (150 g L-') as described previously previously showed (3) that ['4C]sucrose was the major sugar (2, 3). The sections consisted of the pollinated ovules attached to found in the ethanol soluble fraction of kernels grown on labeled approximately a 4 mm thick cob base. Three sections were placed sucrose. With a chase on unlabeled sucrose medium, labeled into each 150 x 50 mm Petri dish and samples were removed reducing sugars accumulated at the expense of sucrose. The for analysis every 5 days through 35 dpp. Kernels were extracted in 5 volumes of 80% ethanol (w:v), observed kinetic pattern of labeling was not consistent with any model that invokes breakdown and resynthesis of the majority filtered through Whatman No. 4, and the filtrate collected for of sucrose in the normal flow of carbon into starch. Recently, soluble carbohydrate determinations. The residue remaining on Schmalstig and Hitz (12) showed that the sucrose analog, 1'- the filter paper was washed twice with ethanol, dried, and used fluorosucrose, could be transported into the maize endosperm. for starch determinations. Sugar Determinations. Two mL of the filtrate were dried and 'Supported in part by the Herman Frash Foundation, Florida Agri- lipids removed by partitioning with hexane and H20 (1: 1, V:V). cultural Experiment Station Journal Series No. 8907. Parts of this work The aqueous phase was dried and the residue derivatized using were taken from a dissertation submitted by B. G. C. in partial fulfillment the methods of Ferguson et aL (5). Changes in sucrose, glucose, and fructose concentrations through development were deterof the Ph.D. degree, University of Florida. ABSTRACT

2 Present address: Department of Horticultural Sciences, Texas A&M

3Abbreviations: SS, sucrose synthase; dpp, days postpollination.

University, College Station, TX 77843. 1219

Plant Physiol. Vol. 88, 1988

COBB AND HANNAH

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FIG. 1. Concentration of sucrose through development in in vitro grown wild-type (left panel), sh2 (middle panel), and shl (right panel) kernels grown with sucrose (0), glucose (A), or fructose (A) as the carbon source.

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FIG. 2. Concentration of glucose through development in in vitro grown wild-type (left panel), sh2 (middle panel), and shl (right panel) kernels grown with sucrose (0), glucose (A), or fructose (U) as the carbon source.

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FIG. 3. Concentration of fructose through development in in vitro grown wild-type (left panel), sh2 (middle panel), and shl (right panel) kernels grown with sucrose (0), glucose (A), or fructose (U) as the carbon source.

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SUCROSE SYNTHESIS IN SHI KERNELS GROWN IN VITRO Table I. Dry Weight, Starch Content and Percentage Germination of shl Kernels Grown in Vitro on Different Sugars Sugar Source

Dry Weight

Starch Content

ercentatge Germination

mg/kernel 93b 125a

% dry weight 52b 40b 60a 76a 107b 46b 63b a,b Significant differences at 5% using Duncan's multiple range test. Sucrose Glucose Fructose

mined by GLC using a Hewlett Packard 5710A gas chromatograph as described previously (2). Starch Determinations. One hundred mg of the dried residue was solubilized in approximately 10 mL of 0.5 N NaOH. After addition of 70 mL of water the pH was adjusted to 4.5 with acetic acid and the volume brought to 100 mL. Starch was determined as free glucose after digestion with amyloglucosidase as described (3). Analysis of Mature Kernels. Kernels were also collected after 35 dpp for analysis of starch content and germination. Kernels were removed from the Petri dishes and air dried. After weighing, kernels were placed in rolled paper towels, wetted with distilled water, and germinated in the dark at 26C for 1 week for

determination of germination. RESULTS Carbohydrate Concentration through Development. The concentration of sucrose, glucose, and fructose in wild type, sh2 and shl kernels grown in vitro on sucrose, glucose, or fructose are shown in Figures 1 through 3. Sucrose was present in the endosperms of all treatments indicating that even maize endosperm deficient in the predominant form of sucrose synthase has the capacity to synthesize sucrose (Fig. 1). Regardless of the carbon source, sucrose accumulated to a higher concentration in the endosperms of shl and sh2, than in wild type. This is also characteristic of these mutants (2, 10) when kernels are grown in the field, and is considered to be due to the disruption of starch synthesis resulting in sucrose accumulation. The concentration of reducing sugars was altered by carbon source to a greater extent than was sucrose (Figs. 2 and 3). Kernels grown on glucose had 2 to 3 times the concentration of glucose, and less fructose, than kernels grown on sucrose (Fig. 2). Similarly, kernels grown on fructose had elevated fructose levels and reduced glucose levels compared to kernels grown on sucrose (Fig. 3). Kernel Growth. Mature viable shl kernels were produced on all carbohydrate sources (Table I). Between 25 and 30 dpp kernels produced on all carbon sources began to exhibit denting of the crown. All mature shl kernels exhibited this phenotype and had the ability to germinate. Kernels of shl grown on glucose had significantly greater kernel weight and starch content than did kernels grown on sucrose (Table I). Percentage of germination of shl kernel grown on glucose was also greater than that of the other sugar sources (Table I). DISCUSSION Wild-type, sh2, and shl kernels were capable of synthesizing sucrose when grown on reducing sugars (Fig. 1). The fact that shl kernels synthesized sucrose when grown on reducing sugars indicates that the major endosperm sucrose synthase encoded by sh/l is not essential for sucrose synthesis in the maize endosperm (Fig. 1). This finding supports the view that SS is involved in starch synthesis and that the most prominent physiological function of SS in the starch biosynthetic pathway is to break down

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sucrose. Even though the reaction catalyzed by SS is highly reversible, there is ample physiological and genetic evidence to support this notion. Analysis of interallelic complementing heterozygotes of normal shl alleles suggests a role for this enzyme in sucrose degradation. Recovery of the wild-type phenotype in these complementing heterozygotes is associated with an increased activity of SS in the direction of sucrose cleavage, but not in the direction of sucrose synthesis (1). SS is found in abundance in maize endosperm, a tissue whose function is to convert sucrose into starch (16). The activity profile of SS corresponds to that for other starch synthetic enzymes (1 1, 16). In addition, mutants deficient in SS are deficient in starch (2) suggesting that the enzyme plays a role in starch synthesis. Previously, we reported that wild type and sh2 kernels would develop normally and produce viable seeds when grown on reducing sugars. However, unlike wild type and sh2 kernels grown in vitro (3), sucrose was not the superior carbon source for shl kernels. These findings also support the view that SS is important in starch metabolism and, specifically, sucrose degradation. If SS metabolizes sucrose as a first step in starch metabolism, then the absence of SS would certainly affect starch synthesis. By supplying reducing sugars to shl kernels the block in sucrose metabolism may be partially bypassed, allowing reducing sugars to be utilized for starch biosynthesis and resulting in kernels having greater starch content. While this is the case for kernels grown on glucose, kernels grown on fructose did not have significantly greater weight or starch content than kernels grown on sucrose. Griffith et al. (7) also found that radioactive glucose was incorporated at a faster rate than fructose in the maize endosperm. The reason for this is not known but it is possible that the enzymic conversion of glucose into an intermediate for starch biosynthesis occurs more efficiently than the conversion of fructose. LITERATURE CITED 1. CHOUREY PS, OE NELSON 1979 Interallelic complementation at the sh locus in maize at the enzyme level. Genetics 91: 317-325 2. COBB GB, LC HANNAH 1983 Development of wild type, shrunken-i and shrunken-2 maize seeds grown in vitro. Theor Appl Genet 65: 47-51 3. COBB BG, LC HANNAH 1986 Sugar utilization by developing wild type and shrunken-2 maize kernels. Plant Physiol 80: 609-61 1 4. FELKER FC, JC SHANNON 1980 Movement of ('4C)-labeled assimilates into kernels of Zea mays L. III. An anatomical examination and microradiographic study of assimilate transfer. Plant Physiol 65: 864-870 5. FERGUSON JE, DB DICKINSON, AM RHODES 1979 Analysis of endosperm sugars in a sweet corn inbred which contains the sugary enhancer gene and comparison of se with other corn genotypes. Plant Physiol 63: 416-420 6. GENGENBACH BG 1977 Development of maize caryopses resulting from in vitro pollination. Planta 134: 91-93 7. GRIFFrrH SM, RJ JONES, ML BRENNER 1987 In vitro sugar transport in Zea mays L. kernels. I. Characteristics of sugar absorption and metabolism by developing maize endosperm. Plant Physiol 84: 467-471 8. JENNER CF 1973 The uptake of sucrose and its conversion to starch in detached ears of wheat. J Exp Bot 24: 295-296 9. JENNER CF 1974 An investigation of the association between the hydrolysis of sucrose and its absorption by grains of wheat. Aust J Plant Physiol 1: 319329 10. LAUGHNAN JR 1953 The effects of the Sh2 factor on carbohydrate reserves in the mature endosperm of maize. Genetics 38: 485-499 11. Ou-LEE T-M, TL SETTER 1985 Enzyme activities of starch and sucrose pathways and growth of apical and basal maize kernels. Plant Physiol 79: 848851 12. SCHMALSTIG JG, WD HITZ 1987 Transport and metabolism of a sucrose analog (1 '-fluorosucrose) into Zea mays L. endosperm without invertase hydrolysis. Plant Physiol 85: 902-905 13. SHANNON JC 1968 Carbon-14 distribution in carbohydrates of immature Zea mays kernels following 14C CO2 treatment of intact plants. Plant Physiol 43: 12 15-1220 14. SHANNON JC 1972 Movement of ["C]-labeled assimilates into kernels of Zea mays L. I. Pattern and rate of sugar movement. Plant Physiol 49: 198-202 15. SHANNON JC, C DoUGHERTY 1972 Movement of 14C labeled assimilates into kernels of Zea mays L. Plant Physiol 49: 203-206 16. TSAI CY, F SALAMINI, OE NELSON 1970 Enzymes of carbohydrate metabolism in the developing maize endosperm. Plant Physiol 46: 299-306