Distribution and Redistribution of Sulfur Supplied as [35S]Sulfate to Roots during Vegetative Growth of Soybean

Soybean (Glycine max L.) plants were grown in nutrient solution containing 10 [mu]M sulfate and were treated at various times with [35S]sulfate for 48 h. Growth was then continued in unlabeled solution. The sulfur content of each leaf increased rapidly until it was about 40% expanded; small, additional increases occurred until the leaf was about 70% expanded after which the sulfur content decreased by about 50%. Leaves that were about 60 to 70% expanded during the pulse were strongly labeled but then underwent a significant loss of 35S label. Leaves that were in the early stages of expansion imported little 35S label during the pulse but acquired 35S label during the chase period as they expanded (i.e. redistribution). Most of the redistributed 35S label was derived from other leaves. The rates of both sulfur import and sulfur export by a leaf were greatest at about 70% expansion. Leaves that acquired 35S label during early development retained a much higher proportion of their label than leaves that were more developed, suggesting that the sulfur acquired by leaves during early development is preferentially incorporated into a pool that is less mobile than the sulfur acquired in the later stages of leaf growth.     

Allocation of S in Generative Growth of Soybean

Soybean plants (Glycine max L. Merr) were grown with 100 [mu]M S and 15 mM N and studied with respect to S allocation during grain development. The grains accounted for 87% of the S taken up after d 42, the balance coming from internal redistribution of S from leaves and pods. Detailed studies of the leaves, pods, and grains associated with leaf axils 6 and 7 showed that sulfate accumulated in the pods as they expanded to 50% of full length, ahead of grain enlargement, but declined to very low levels as grain growth commenced. Conversely, homoglutathione (hGSH), cysteine, and methionine increased. In developing grains, hGSH accounted for 60 to 90% of the soluble-S but sulfate was barely detectable. The data are consistent with a model in which, under S-limiting conditions, the pods act as sinks for sulfate and grain growth initiates the assimilation of sulfate into hGSH in the pods, and then into developing grains, where it is incorporated into grain proteins.     

Mobilization of sulphur in soybean cotyledons during germination

Soybean seeds ( Glycine max L. cv , Stephens) contain a large amount of sulphur (ca 40 μ mol seed⁻¹), mostly in the insoluble fraction in the cotyledons. During germination in nutrient solution lacking sulphur the amount of insoluble sulphur decreases to very low levels. This is accompanied by a transitory increase in the pool of soluble sulphur which then declines. All of the sulphur lost from the cotyledons is quantitatively recovered in the seedling. In the short term, the root and the stem are the most important sinks for sulphur from the cotyledons but as growth proceeds the shoot becomes the dominant sink for remobilized sulphur. Within the shoot most of the sulphur is recovered in leaves L1 and L2. The growth of L3 and, to a lesser extent, L2, was retarded due to sulphur insufficiency. The cotyledons of plants treated with 20 μ M sulphate also exhibited mobilization of sulphur from the insoluble fraction except that the maximum rate of loss of sulphur occurred somewhat later. Plants grown with sulphate exhibited a net gain of sulphur and did not exhibit sulphur insufficiency. In these plants, endogenous sulphur from the cotyledons was directed into L1–L3 and this sulphur remained within these leaves for the duration of the experiment. The delivery of exogenous sulphur (supplied as [³⁵S]sulphate via the roots) to the leaves increased with leaf number. In leaves L1–L3, the level of exogenous sulphur in any one leaf declined with time, indicating that this sulphur was remobilized and did not mix with the sulphur derived from the cotyledons. It was concluded that the cotyledons are an important source of sulphur to support early plant growth and development of soybean.     

Effect of Nitrogen Nutrition on Remobilization of Protein Sulfur in the Leaves of Vegetative Soybean and Associated Changes in Soluble Sulfur Metabolites

The hypothesis that protein S is remobilized from mature leaves in response to N stress but not S stress was examined by transferring vegetative soybean (Glycine max L. Merr) plants grown with adequate sulfate and nitrate to nutrient medium with low sulfate (5 [mu]M) and nitrate at either 15, 7.5, 2, or 0.25 mM. Soluble S decreased to very low levels in mature and maturing leaves, especially in low-N plants. At high [N], insoluble S (protein) in mature leaves remained constant, but at low [N], after the soluble S declined, up to 40% of the insoluble S was exported. The losses were complemented by gains, initially in soluble S, but subsequently in insoluble S, in the expanding leaves and the root. In low-N plants, but not in high-N plants, the decrease in insoluble S in mature leaves was complemented by increases in homoglutathione (hGSH), Cys, and Met. At low [N], but not at high [N], the developing leaf, leaf 5, contained high amounts of soluble S, mostly hGSH. The results suggest that, at low [N], protein S is metabolized to hGSH, which serves as the principal transport compound for the export of organic S.     

Kappaphycus malesianus sp. nov.: a new species of Kappaphycus (Gigartinales,Rhodophyta) from Southeast Asia

A new species, Kappaphycus malesianus, is established as a new member of the genus Kappaphycus. Locally known as the “Aring-aring” variety by farmers in Malaysia and the Philippines, this variety has been commercially cultivated, often together with Kappaphycus alvarezii due to the similarities in morphology. Despite also producing kappa-carrageenan, the lower biomass of the K. malesianus when mixed with K. alvarezii ultimately affects the carrageenan yield. Morphological observations, on both wild and cultivated plants, coupled with molecular data have shown K. malesianus to be genetically distinct from its Kappaphycus congeners. The present study describes the morphology and anatomy of this new species as supported by DNA data, with additional morphological features for distinguishing between commercial Kappaphycus cultivars.     

Genetic diversity of Kappaphycus Doty and Eucheuma J. Agardh (Solieriaceae,Rhodophyta) in Southeast Asia

The commercial importance of carrageenophytes Kappaphycus and Eucheuma is well known, with much interest in terms of cultivation, marketing, and research. Considering the many lucrative prospects, these red seaweeds were introduced into various parts of the world for farming, where merely a few were comprehensively documented. Despite being extensively cultivated throughout Southeast Asia, the genetic diversity of Kappaphycus and Eucheuma is poorly studied, where heavy reliance is placed on the use of local or commercial names for identifications. This study used the mitochondrial-encoded cox1 and cox2–3 spacer genetic markers to investigate the Kappaphycus and Eucheuma haplotypes, cultivated and wild, available throughout Southeast Asia. Concatenated cox1–cox2–3 spacer datasets were also analyzed. The near full-length cox1 gene is preferred at revealing the genetic diversity of Kappaphycus and Eucheuma, provided a larger reference database is available. Both molecular markers were capable of delineating common members of the genus Kappaphycus (i.e., Kappaphycus alvarezii, Kappaphycus striatus, and Kappaphycus cottonii) and Eucheuma denticulatum, and revealed interesting genotypes and new species which may be potential alternatives to the common cultivars as well as materials for research. The relative scarcity of Eucheuma species is discussed and future sites for sampling are recommended.     

Assessment of Four Molecular Markers as Potential DNA Barcodes for Red Algae Kappaphycus Doty and Eucheuma J. Agardh (Solieriaceae,Rhodophyta)

DNA barcoding has been a major advancement in the field of taxonomy, seeing much effort put into the barcoding of wide taxa of organisms, macro and microalgae included. The mitochondrial-encoded cox1 and plastid-encoded rbcL has been proposed as potential DNA barcodes for rhodophytes, but are yet to be tested on the commercially important carrageenophytes Kappaphycus and Eucheuma. This study gauges the effectiveness of four markers, namely the mitochondrial cox1, cox2, cox2-3 spacer and the plastid rbcL in DNA barcoding on selected Kappaphycus and Eucheuma from Southeast Asia. Marker assessments were performed using established distance and tree-based identification criteria from earlier studies. Barcoding patterns on a larger scale were simulated by empirically testing on the commonly used cox2-3 spacer. The phylogeny of these rhodophytes was also briefly described. In this study, the cox2 marker which satisfies the prerequisites of DNA barcodes was found to exhibit moderately high interspecific divergences with no intraspecific variations, thus a promising marker for the DNA barcoding of Kappaphycus and Eucheuma. However, the already extensively used cox2-3 spacer was deemed to be in overall more appropriate as a DNA barcode for these two genera. On a wider scale, cox1 and rbcL were still better DNA barcodes across the rhodophyte taxa when practicality and cost-efficiency were taken into account. The phylogeny of Kappaphycus and Eucheuma were generally similar to those earlier reported. Still, the application of DNA barcoding has demonstrated our relatively poor taxonomic comprehension of these seaweeds, thus suggesting more in-depth efforts in taxonomic restructuring as well as establishment.     

Effect of Sulfur Nutrition on the Redistribution of Sulfur in Vegetative Soybean Plants

Soybean (Glycine max L.) plants were grown with sulfate at 2 (S2) or 20 [mu]M (S20) and treated with [35S]sulfate between d 36 and 38. Growth was continued with or without 20 [mu]M sulfate (i.e. S2 -> S0, S2 -> S20, etc.). When the leaves of S20 -> S20 plants were 70% expanded, they exported S and 35S label from the soluble fraction, largely as sulfate, to new expanding leaves. However, 35S label in the insoluble fraction was not remobilized. Very little of the 35S label in the soluble fraction of the leaves of S20 -> S0 plants was redistributed; most was incorporated into the insoluble fraction. The low levels of S remobilization from the insoluble fraction were attributed to the high level of N in the nutrient solution (15 mM). Most of the 35S label in S2 plants at d 38 occurred in the soluble fraction of the roots. In S2 -> S0 plants the 35S label was incorporated into the insoluble fraction of the roots, but in S2 -> S20 plants 35S label was rapidly exported to leaves 3 to 6. It was concluded that the soluble fraction of roots contains a small metabolically active pool of S and another larger pool that is in slow equilibrium with the small pool.     

Inhibition of sulphur redistribution into new leaves of vegetative soybean by excision of the maturing leaf

When soybean plants are pulsed with [³⁵S]sulphate, label is subsequently redistributed from the roots to the leaves. This confounds studies to measure the redistribution of label from leaves. Accordingly, soybean plants ( Glycine max [L.] Merr. cv. Stephens) were grown in 20 μ M sulphate and a small portion of the root system (donor root) was pulsed with [³⁵S]sulphate for 24 h. After removing the donor root, the plants were transferred into unlabelled solution, either without sulphate (S20→SO) or with 20 μ M sulphate (S20→20) (intact plants). Also at this time, the expanding leaf (L3) was excised from half of the plants in each treatment (excised plants). Immediately after the pulse, only ca 15% of the label occurred in the roots and ca 40% in the expanding leaf, L3, mostly in the soluble fraction. In intact S20→20 plants, ³⁵S-label was exported from the soluble fraction of L3, mostly as sulphate, whilst L4 and L5 imported label. Similar responses occurred in S20→SO plants except that export of label from L3 was more rapid. Excision of L3 from S20→S20 plants inhibited labelling of leaves L4-L6 but not total sulphur, whereas in S20→SO plants, excision of L3 inhibited the import of both total sulphur and ³⁵S-label in leaves L4, L5 and L6. The data suggest that the soluble fraction of almost fully expanded leaves is an important reserve of sulphur for redistribution to growing leaves. The ³⁵S-label in the root system exhibited fluctuations consistent with its proposed role in the recycling of soluble sulphur from the leaves.     

Effect of nitrogen nutrition on the export of sulphur from leaves in soybean

Soybean plants were grown in complete solution for 33 days and then transferred to medium containing inadequate sulphur (5 t M) and nitrogen at 15, 7.5, 2 or 0.25 mt M. In mature leaves (L1 and L2), and leaves that were 70% expanded at day 33 (L3), the net loss of sulphur over the ensuing 25 days was inversely related to the level of nitrogen nutrition. Leaf 5, which formed during the study period, exhibited complementary characteristics; the increase in the sulphur content was inversely related to the level of nitrogen nutrition even though low nitrogen nutrition supported less growth. L4, which was 31% expanded at day 33, exhibited intermediate characteristics. 35S-Labelled sulphate was supplied to all of the plants for 48 h at day 31 and was distributed principally to L3 at day 33. During early development, L5 became heavily labelled but, at low nitrogen nutrition, the massive import of total sulphur into L5 during the late stages of development was not accompanied by a commensurate increase in 35S-label, indicating that redistribution of soluble sulphur from mature leaves was not involved. The loss of sulphur from mature leaves was parallelled by similar changes in nitrogen at all levels of nitrogen nutrition. Collectively, the data suggest that a common mechanism, presumably proteolysis, is involved in the export of sulphur and nitrogen from mature leaves and that this process is inhibited at high levels of nitrogen nutrition, even under conditions of sulphur deficiency.