"Creating male sterile line by perturbation in carbohydrate metabolismIntroduction: - Carbohydrates were shown to play a critical role in anther and pollendevelopment. They are nutrients used to sustain growth as well as signal to influencedevelopment in vivo and in vitro. Accordingly, different male sterile lines were shown to becharacterized by perturbed carbohydrate metabolism. In accordance with the critical functionof carbohydrate for pollen development and anther function, it was reported that various malesterile plants show a disturbed carbohydrate metabolism. A common effect of the differentmutations characterized by so far seems to be the perturbation of starch accumulation. Inwheat and rice a water deficit during meiosis led to an arrest in pollen development thatcorrelated with alteration in carbohydrate metabolism and a drastic decrease in invertaseactivity. Like-wise a strong correlation between male sterility and invertase function wasdemonstrated for tobacco. Combining the data available on the importance of carbohydratesfor pollen formation and development and the distinctive involvement of invertases incarbohydrate partitioning leads to the conclusion that extracellular cleavage of sucrose byinvertases is critically involved in these processes.Mechanism: - Assimilates are produced in photosynthetically active source tissues andtransported to photosynthetically less active or inactive sink tissues. An unloading pathwayvia the apoplasmic space is mandatory for symplastically isolated cells, such as developingpollen, and also can contribute prominently in other actively growing tissues. Sucrose isreleased from the sieve elements of the phloem into the apoplast via a sucrose transporter. Anextracellular invertase ionically bound to the cell wall irreversibly hydrolyses the transportsugar sucrose. The hexose monomers are taken up into the sink cell by high-affinity hexosetransporters. These key reactions create a localized concentration gradient, thus promotingphloem unloading via an apoplastic pathway and increasing the sink strength of the corresponding sink tissue. The importance of extracellular invertases for assimilatepartitioning and source sink regulation has been suggested in recent years in a number ofstudies and the functional coupling with hexose transporters is supported by a coordinatedregulation . It has been shown that extracellular invertases are encoded by small gene familiesthat show a highly differential sink tissue-specific expression pattern. The identification ofextracellular invertase isoenzymes from tomato, potato and tobacco that are expressed inanther tissues supports a link between extracellular sucrose cleavage and anther and pollendevelopment.Here, we report cloning of a gene encoding an extracellular invertase isoenzyme fromtobacco that shows a specific spatial and temporal expression in anthers. Transgenic tobaccoplants transformed with an antisense construct of extracellular invertase Nin88 under controlof its own promoter are blocked in early pollen development, thus causing male sterility.These results prove the vital importance of extracellular invertase for pollen development bycontributing to the supply of carbohydrates via an apoplastic pathway and metabolicsignalling and offer the possibility to engineer male sterility for hybrid seed production.Inducing male sterility by metabolic engineering: Perturbation of the carbohydratesupply and sugar status during pollen development:- The existence of specific extracellular invertase isoforms in anthers and the importance ofcarbohydrates for pollen formation and development offer a promising starting point toinfluence male fertility by metabolic engineering of the carbohydrate supply to the anthersand pollen. In tobacco an extracellular invertase was identified that shows a very specificspatial and temporal expression pattern. This invertase isoenzyme, Nin88, is expressed in thetapetum early in pollen development, until the tapetum is degraded, and found to be expressesin the pollen grain itself from the tetrad stage on as shown by RNA in situ and immune-localization studies. Cloning of the Nin88 promoter allowed the cell specific reduction of theextracellular invertase activity in these tissues by using an invertase- antisense constructexpressed in transgenic tobacco plant under control of this promoter. The Nin88-antisenseplants were identical to untransformed plant with respect to growth rate, height, morphologyof vegetative and floral organs and tissues, time of flowering, and flower coloration pattern.However a substantial portion of the antisense plant failed to produce seed capsules orshowed a reduced seeds correspond to the germination efficiency of pollen isolated frommature anther in an in vitro germination assay. The plants that were unable to produce seedsshowed a germination efficiency of less than 2%despite the fact that 90% of the pollen wasviable. Analysis of these plants showed a highly tissue specific and significant reduction ofextracellular invertase activity during pollen development. The reduction was mostsignificant at the stage of microspore mitosis I which shows a maximum of invertase activityin wild-type pollen development. This is supported by the finding that developmentallyarrested pollen from the antisense plants are characterized by one nucleus, whereas maturepollen from wild type plant possess two nuclei. Thus the resulting carbohydrate deficiencyduring a critical stage of pollen development leads to a development block at the unicellularmicrospore stage. Further analysis revealed that the inability of pollen from Nin88-antisenseplants to germinate is inversely correlate with the ability to accumulate starch. Pollen fromthe Nin-88-antisense plants is also characterized by a different morphology. Lightmicroscopic inspection revealed that pollen from antisense plants represents a heterogonouspopulation with respect to pollen grains of different size and shape. The proportion of wild- type-like pollen of the various antisense lines corresponds to the germination efficiency. Thedifferences in morphology between wild-type pollen and pollen from antisense lines, inparticular, are evident in analysis with electron microscope. Scanning electron microscope revealed a distorted and invaginated morphology, indicating a reduced amount of intracellularmaterial or turgor.Using in vitro maturation assays, it could be demonstrated that this development can bepartially be overcome by supplying the pollen with external sugars. Both sucrose and hexosetransporters have been found in anthers are assumed to be involved in anther/pollendevelopment. These results indicate that developmental block is not solely due toperturbation of the carbohydrate supply but that the function of extracellular invertase is alsocritical for metabolic signalling required for pollen development. This assumption is backedup by other findings showing special signalling functions for hexose and sucrose. Sugars areknown to be not only nutrients but also signal molecules which like hormones and incombination with hormones and other stimuli, can regulate many aspects of the plant’s lifefrom gene expression to long distance nutrient allocation. A high hexose/sucrose ratiofavours cell division activity, whereas a low hexose/sucrose ration induces storage activity.The reduction of extracellular invertase activity in the pollen of the invertase- antisense plantstherefore determines a low hexose/sucrose ratio interfering with normal regulatory functionand thereby affecting cell division activity and normal development. The data discussedsuggested that tissue specific antisense repression of extracellular invertase in the tapetum hasa dual effect.1. Limiting or abolishing the carbohydrate supply and thus starving the developingpollen for the carbon source, which resulted in the inability to accumulate starch andother intracellular storage material.2. Perturbation of the sugar status thus affecting sugar sensing and signalling required asmetabolic stimulus for regulating pollen development. "