0 1 . 00 / 0 Shrunken-i Encoded Sucrose Synthase Is Not Required for Sucrose Synthesis in the Maize Endosperm 1

Kernels of wild-type maize (Zea mays L.) shrunken-i (shi), deficient in the predominant form of endosperm sucrose synthase and shrunken-2 (sh2), deficient in 95% of the endosperm ADP-glucose pyrophosphorylase were grown in culture on sucrose, glucose, or fructose as the carbon source. Analysis of the endosperm extracts by gas-liquid chromatography revealed that sucrose was present in the endosperms of all genotypes, regardless of carbon supply, indicating that all three genotypes are capable of synthesizing sucrose from reducing sugars. The finding that sucrose was present in shl kernels grown on reducing sugars is evidence that shrunken-i encoded sucrose synthase is not necessary for sucrose synthesis. Shrunken-i kernels developed to maturity and produced viable seeds on all carbon sources, but unlike wild-type and sh2 kernels grown in vitro, sucrose was not the superior carbon source. This latter result provides further evidence that the role of sucrose synthase in maize endosperm is primarily that of sucrose degradation. In cereals, the metabolic machinery involved in utilizing the hexoses of sucrose which enters the base of the seed and ultimately converts that carbon into starch in the endosperm has been the subject of many investigations. In attempts to identify and order the intermediates involved in this long and complicated pathway, studies have recently focused on sucrose and the 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 the seed, it still remains an open question whether sucrose is resynthesized from C-6 sugars in the process of starch synthesis. Jenner (8, 9) using asymmetrically labeled sucrose, concluded that sucrose was not broken down and resynthesized in the developing wheat kernel. In the case of maize endosperm we previously showed (3) that ['4C]sucrose was the major sugar found in the ethanol soluble fraction of kernels grown on labeled sucrose. With a chase on unlabeled sucrose medium, labeled reducing sugars accumulated at the expense of sucrose. The observed kinetic pattern of labeling was not consistent with any model that invokes breakdown and resynthesis of the majority of sucrose in the normal flow of carbon into starch. Recently, Schmalstig and Hitz (12) showed that the sucrose analog, 1'fluorosucrose, could be transported into the maize endosperm. 'Supported in part by the Herman Frash Foundation, Florida Agricultural Experiment Station Journal Series No. 8907. Parts of this work were taken from a dissertation submitted by B. G. C. in partial fulfillment of the Ph.D. degree, University of Florida. 2 Present address: Department of Horticultural Sciences, Texas A&M University, College Station, TX 77843. Since this molecule is not hydrolyzed by invertase this suggests that hydrolysis of sucrose is not a requirement for uptake. Perhaps unexpectedly, maize kernels have the ability to grow and develop into viable seeds when sucrose is replaced with reducing sugars (3). This observation clearly shows that sucrose is not essential for kernel development and raises the possibility that there is some interconversion of the various sugars that come early in the metabolic path. Consistent with this are the earlier observations of Shannon and coworkers (4, 13-15) who showed that sucrose can be broken down upon entry into the maize seed. Their data further suggest that sucrose may be resynthesized in the endosperm cells before entry into the starch biosynthetic pathway. By use of the in vitro kernel development scheme of Gengenbach (6) as modified by us (2) we can ask very simple and directly whether kernels have the ability to synthesize sucrose when they 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 necessary for the observed conversion. Indeed, data presented here support the view that the physiological role ofSS is predominantly that of sucrose degradation. MATERIALS AND METHODS Plant Material. Ears of shl, sh2, and wild-type maize (Zea mays L.) were hand pollinated and harvested at 5 dpp. These were transported on ice and held at 4°C until processed. The outer husks were removed, the ear sprayed with ethanol and flamed. Ear sections, consisting of 2 rows of 8 kernels, were removed and placed in media containing sucrose (150 g L'), glucose (150 g L`), or fructose (150 g L-') as described previously (2, 3). The sections consisted ofthe pollinated ovules attached to approximately a 4 mm thick cob base. Three sections were placed into each 150 x 50 mm Petri dish and samples were removed for analysis every 5 days through 35 dpp. Kernels were extracted in 5 volumes of 80% ethanol (w:v), filtered through Whatman No. 4, and the filtrate collected for soluble carbohydrate determinations. The residue remaining on the filter paper was washed twice with ethanol, dried, and used for starch determinations. Sugar Determinations. Two mL of the filtrate were dried and lipids removed by partitioning with hexane and H20 (1: 1, V:V). The aqueous phase was dried and the residue derivatized using the methods of Ferguson et aL (5). Changes in sucrose, glucose, and fructose concentrations through development were deter3Abbreviations: SS, sucrose synthase; dpp, days postpollination.