Cause Of Male Sterility Research Articles

Marker assisted selection reveal SNP deletion mutation in gene associated with cytoplasmic male sterility in Solanumvillosum

Male sterility (MS) is powerful tool for improvement of various traits of economic importance. MS has not been characterized in Solanum villosum, and molecular mechanism underlying mutation change have not yet been studied. In this study, Sanger sequence platform was employed to amplify the MS genic region using mt ATPase marker. The molecular mechanism governing sterility was studied, and the results detected the amplification region at 1150bp. The sequence query was 97 % identical with atp subunit 6 gene loci. Significance mutation detected in this study was deletion GG (delGG) and deletion C (delC) occurring between 536bp and 542bp position in atp6 gene transcript. The mutation score was 16 for delGG and 17 for delC, with a 90 % mutation confidence interval. The study observed that SNP changes due to base deletions in the atp6 gene region were the main cause of male sterility in the studied mutants due to anticipated frameshifts of the open reading frame (ORF) of the mitochondrial atp6 gene, this is regarded as cytoplasmic male sterility type (CMS). Deformed and low pollen count was morphological changes confirmed this study that associated with sterility. Disruption of the ATP synthesis in mitochondria, limits the energy supply for pollen grain formation and anticipated pollen sterility however, mitochondria nuclear gene interaction governing the CMS should be further studied in case of S.villosum. These findings shed-light on molecular mechanism underlying the CMS and can be utilized as a molecular marker for agronomic traits improvement of S.villosum.

ADAD2 functions in spermiogenesis and piRNA biogenesis in mice.

Adenosine deaminase domain containing 2 (ADAD2) is a testis-specific protein composed of a double-stranded RNA binding domain and a non-catalytic adenosine deaminase domain. A recent study showed that ADAD2 is indispensable for the male reproduction in mice. However, the detailed functions of ADAD2 remain elusive. This study aimed to investigate the cause of male sterility in Adad2 mutant mice and to understand the molecular functions of ADAD2. Adad2 homozygous mutant mouse lines, Adad2-/- and Adad2Δ/Δ , were generated by CRISPR/Cas9. Western blotting and immunohistochemistry were used to reveal the expression and subcellular localization of ADAD2. Co-immunoprecipitation tandem mass spectrometry was employed to determine the ADAD2-interacting proteins in mouse testes. RNA-sequencing analyses were carried out to analyze the transcriptome and PIWI-interacting RNA (piRNA) populations in wildtype and Adad2 mutant testes. Adad2-/- and Adad2Δ/Δ mice exhibit male-specific sterility because of abnormal spermiogenesis. ADAD2 interacts with multiple RNA-binding proteins involved in piRNA biogenesis, including MILI, MIWI, RNF17, and YTHDC2. ADAD2 co-localizes and forms novel granules with RNF17 in spermatocytes. Ablation of ADAD2 impairs the formation of RNF17 granules, decreases the number of cluster-derived pachytene piRNAs, and increases expression of ping-pong-derived piRNAs. In collaboration with RNF17 and other RNA-binding proteins in spermatocytes, ADAD2 directly or indirectly functions in piRNA biogenesis.

Transcriptomic analysis reveals candidate genes for male sterility in Prunus sibirica.

BackgroundThe phenomenon of male sterility widely occurs in Prunus sibirica and has a serious negative impact on yield. We identified the key stage and cause of male sterility and found differentially expressed genes related to male sterility in Prunus sibirica, and we analyzed the expression pattern of these genes. This work aimed to provide valuable reference and theoretical basis for the study of reproductive development and the mechanisms of male sterility in Prunus sibirica.MethodThe microstructures of male sterile flower buds and male fertile flower buds were observed by paraffin section. Transcriptome sequencing was used to screen genes related to male sterility in Prunus sibirica. Quantitative real-time PCR analysis was performed to verify the transcriptome data.ResultsAnther development was divided into the sporogenous cell stage, tetrad stage, microspore stage, and pollen maturity stage. Compared with male fertile flower buds, in the microspore stage, the pollen sac wall tissue in the male sterile flower buds showed no signs of degeneration. In the pollen maturity stage, the tapetum and middle layer were not fully degraded, and anther development stopped. Therefore, the microspore stage was the key stage for anther abortion , and the pollen maturity stage was the post stage for anther abortion. A total of 4,108 differentially expressed genes were identified by transcriptome analysis. Among them, 1,899 were up-regulated, and 2,209 were down-regulated in the transcriptome of male sterile flower buds. We found that “protein kinase activity”, “apoptosis process”, “calcium binding”, “cell death”, “cytochrome c oxidase activity”, “aspartate peptidase activity”, “cysteine peptidase activity” and other biological pathways such as “starch and sucrose metabolism” and “proteasome” were closely related to male sterility in Prunus sibirica. A total of 331 key genes were preliminarily screened.ConclusionThe occurrence of male sterility in Prunus sibirica involved many biological processes and metabolic pathways. According to the results of microstructure observations, related physiological indexes determination and transcriptome analysis, we reveal that the occurrence of male sterility in Prunus sibirica may be caused by abnormal metabolic processes such as the release of cytochrome c in the male sterile flower buds, the imbalance of the antioxidant system being destroyed, and the inability of macromolecular substances such as starch to be converted into soluble small molecules at the correct stage of reproductive development, resulting in energy loss. As a result, the tapetum cannot be fully degraded, thereby blocking anther development, which eventually led to the formation of male sterility.

Comparative analysis of morphology, cytology, and mitochondrial transcriptome of sterile male flowers of rubber tree

AbstractRubber tree [Hevea brasiliensis (Willd. ex A. Juss.) Müll. Arg.] is the exclusive commercial source of natural rubber. Male‐sterile (MS) germplasm has proven to be valuable for rubber tree hybrid breeding, but its male sterility mechanism remains unclear. Here, morphological observations of a new MS clone Reyan93‐114 showed shriveled anthers, short filaments, and withered male flowers. Transmission electron microscopy revealed the tapetal cells of the anthers were hypertrophic and vacuolated. Microspore protoplasts reduced in size, and the microspore wall had obvious defects: the incomplete intine and the insufficiently deposited exine. Organelles had disappeared in both tapetal cells and microspores. Comparative mitochondrial transcriptome analysis identified three differentially expressed genes in the MS clones: orf126, orf51, and atp8. In particular, orf126 and orf51 were unique insertions in the MS mitochondrial genomes. The former was an unknown open reading frame, and the latter was a fusion gene that contained a portion of atp9. These two genes should be studied as candidate male sterility genes in rubber tree. These results clarified the cytological causes of male sterility and expanded to understand the relationship between mitochondrial genes and male sterility in rubber tree.

Chakras and energy alterations in patients with oligospermia

Oligospermia is a male fertility problem characterized by a low sperm count. In Western Medicine, the causes of male sterility are mostly unknown. According to Traditional Chinese Medicine (TCM), oligospermia stems from Kidney deficiency.

Usp14 is required for spermatogenesis and ubiquitin stress responses in Drosophila melanogaster.

Deubiquitylating (DUB) enzymes free covalently linked ubiquitin moieties from ubiquitin-ubiquitin and ubiquitin-protein conjugates, and thereby maintain the equilibrium between free and conjugated ubiquitin moieties and regulate ubiquitin-mediated cellular processes. Here, we performed genetic analyses of mutant phenotypes in Drosophila melanogaster and demonstrate that loss of Usp14 function results in male sterility, with defects in spermatid individualization and reduced testicular free monoubiquitin levels. These phenotypes were rescued by germline-specific overexpression of wild-type Usp14. Synergistic genetic interactions with Ubi-p63E and cycloheximide sensitivity suggest that ubiquitin shortage is a primary cause of male sterility. In addition, Usp14 is predominantly expressed in testes in Drosophila, indicating a higher demand for this DUB in testes that is also reflected by testis-specific loss-of-function Usp14 phenotypes. Collectively, these results suggest a major role of Usp14 in maintaining normal steady state free monoubiquitin levels during the later stages of Drosophila spermatogenesis.This article has an associated First Person interview with the first author of the paper.

Mutations in TTC29, Encoding an Evolutionarily Conserved Axonemal Protein, Result in Asthenozoospermia and Male Infertility

In humans, structural or functional defects of the sperm flagellum induce asthenozoospermia, which accounts for the main sperm defect encountered in infertile men. Herein we focused on morphological abnormalities of the sperm flagellum (MMAF), a phenotype also termed "short tails," which constitutes one of the most severe sperm morphological defects resulting in asthenozoospermia. In previous work based on whole-exome sequencing of a cohort of 167 MMAF-affected individuals, we identified bi-allelic loss-of-function mutations in more than 30% of the tested subjects. In this study, we further analyzed this cohort and identified five individuals with homozygous truncating variants in TTC29, a gene preferentially and highly expressed in the testis, and encoding a tetratricopeptide repeat-containing protein related to the intraflagellar transport (IFT). One individual carried a frameshift variant, another one carried a homozygous stop-gain variant, and three carried the same splicing variant affecting a consensus donor site. The deleterious effect of this last variant was confirmed on the corresponding transcript and protein product. In addition, we produced and analyzed TTC29 loss-of-function models in the flagellated protist T.brucei and in M.musculus. Both models confirmed the importance of TTC29 for flagellar beating. We showed that in T.brucei the TPR structural motifs, highly conserved between the studied orthologs, are critical for TTC29 axonemal localization and flagellar beating. Overall our work demonstrates that TTC29 is a conserved axonemal protein required for flagellar structure and beating and that TTC29 mutations are a cause of male sterility due to MMAF.

Comparative studies of live tapetum cells in sterile garlic (Allium sativum) and fertile leek (Allium ampeloprasum) using the fluorescence lifetime imaging analytical method

Commercially cultivated Allium sativum has lost the ability to reproduce sexually due to various developmental abnormalities mainly in the male line associated with a phenomenon called male sterility. Numerous studies focused on discovering the cause of garlic male sterility at the tissue and cellular level have not yet fully resolved this issue. Here, we have focused our attention on the tapetum, i.e. a nutritive tissue regarded in plants as a critical factor for formation of viable pollen. We have applied a novel method, Fluorescence Lifetime Imaging (FLIM), which represents one of the most sensitive biophysical tools for assessment of changes in fluorophore lifetime, depending on its metabolic state in a live cell. Contrary to traditional methods based on cell fixation, FLIM analysis provides live tapetal cell imaging, which facilitates monitoring metabolic changes in the cell. We have shown qualitative metabolic discrepancies in live cells of the tapetum in sterile garlic and fertile leek and proposed a correlative metabolic model of microspore and tapetum development in garlic cultivars, indicating that the disturbances in the temporal coordination of programmed cell death within the anther might be regarded as the main cause of male sterility in the analysed A. sativum cultivars.

Involvement of a universal amino acid synthesis impediment in cytoplasmic male sterility in pepper.

To explore the mechanisms of pepper (Capsicum annuum L.) cytoplasmic male sterility (CMS), we studied the different maturation processes of sterile and fertile pepper anthers. A paraffin section analysis of the sterile anthers indicated an abnormality of the tapetal layer and an over-vacuolization of the cells. The quantitative proteomics results showed that the expression of histidinol dehydrogenase (HDH), dihydroxy-acid dehydratase (DAD), aspartate aminotransferase (ATAAT), cysteine synthase (CS), delta-1-pyrroline-5-carboxylate synthase (P5CS), and glutamate synthetase (GS) in the amino acid synthesis pathway decreased by more than 1.5-fold. Furthermore, the mRNA and protein expression levels of DAD, ATAAT, CS and P5CS showed a 2- to 16-fold increase in the maintainer line anthers. We also found that most of the amino acid content levels decreased to varying degrees during the anther tapetum period of the sterile line, whereas these levels increased in the maintainer line. The results of our study indicate that during pepper anther development, changes in amino acid synthesis are significant and accompany abnormal tapetum maturity, which is most likely an important cause of male sterility in pepper.

Activity of selected hydrolytic enzymes in Allium sativum L. anthers

The aim of the study was to determine enzymatic activity in sterile Allium sativum anthers in the final stages of male gametophyte development (the stages of tetrads and free microspores). The analysed enzymes were shown to occur in the form of numerous isoforms. In the tetrad stage, esterase activity was predominant, which was manifested by the greater number of isoforms of the enzyme. In turn, in the microspore stage, higher numbers of isoforms of acid phosphatases and proteases were detected. The development of sterile pollen grains in garlic is associated with a high level of protease and acid phosphatase activity and lower level of esterase activities in the anther locule. Probably this is the first description of the enzymes activity (ACPH, EST, PRO) in the consecutives stages of cell wall formation which is considered to be one of the causes of male sterility in flowering plant.

Defective callose walls and cell plates during abnormal meiosis cause male-sterility in the oat mutant zbs1

AbstractDuring meiosis in flowering plants, degradation of the callose wall in tetrads releases newly produced microspores, which develop into mature pollen grains. In this study, we identified zbs1, a male-sterile mutant of naked oat (Avena nuda L.) that displayed complete spikelet sterility due to inviable mature pollen. The abnormal pollen grains originated from microspores with a defective callose wall and cell plate during meiosis. The defective callose wall and cell plate of the zbs1 mutant were detected by the labeling of cell wall epitopes (β-1,3-glucan) with immunogold during meiosis, and an abnormal chromosome configuration was observed by propidium iodide staining. The mature pollen grains of the zbs1 mutant were irregular in shape, and abnormal germination was observed by scanning electron microscopy. Together, our results indicate that the cause of male sterility in zbs1 is abnormal meiosis.

Phenotypic, cytogenetic and spike fertility characterization of a population of male-sterile triticale

Triticale (X Triticosecale Wittmack) is a good cereal for production of flour and feed. A segregating population of triticale was developed from a male-sterile (MS) plant. To determine whether this new source of male sterility in triticale is appropriate for use in breeding programs the expression of the male sterility phenotype was characterized through spike fertility, meiotic behavior, and pollen. Controlled crosses between male-sterile plants and control varieties male-fertile (MF) of triticale were also conducted, and cytological analyses were performed in the F2 and backcross plants. Plants with male-sterile phenotypes displayed reduced spike fertility when compared to plants with male-fertile phenotypes. Compared to male-fertile plants, male-sterile plants exhibited a lower percentage of normal meiotic cells, a reduced meiotic index and reduced pollen viability. The F2 plants had improved pollen fertility when compared to the male-sterile population; however there were no corresponding improvements in the percentage of normal meiotic cells or in the meiotic index. A single generation of backcrosses resulted in an improved meiotic index and increased pollen viability. However, no changes in the percentage of normal meiotic cells were observed. Meiotic instability, which was shown to be inheritable, was the likely cause of male sterility. Therefore, the use of this population in triticale breeding was considered to be inappropriate because it could promote or contribute to the maintenance of meiotic instability, which is commonly observed in this species.

Melav2, an elav-like gene, is essential for spermatid differentiation in the flatworm Macrostomum lignano

BackgroundFailure of sperm differentiation is one of the major causes of male sterility. During spermiogenesis, spermatids undergo a complex metamorphosis, including chromatin condensation and cell elongation. Although the resulting sperm morphology and property can vary depending on the species, these processes are fundamental in many organisms. Studying genes involved in such processes can thus provide important information for a better understanding of spermatogenesis, which might be universally applied to many other organisms.ResultsIn a screen for genes that have gonad-specific expression we isolated an elav-like gene, melav2, from Macrostomum lignano, containing the three RNA recognition motifs characteristic of elav-like genes. We found that melav2 mRNA was expressed exclusively in the testis, as opposed to the known elav genes, which are expressed in the nervous system. The RNAi phenotype of melav2 was characterized by an aberrant spermatid morphology, where sperm elongation often failed, and an empty seminal vesicle. Melav2 RNAi treated worms were thus male-sterile. Further analysis revealed that in melav2 RNAi treated worms precocious chromatin condensation occurred during spermatid differentiation, resulting in an abnormally tightly condensed chromatin and large vacuoles in round spermatids. In addition, immunostaining using an early-spermatid specific antibody revealed that melav2 RNAi treated worms had a larger amount of signal positive cells, suggesting that many cells failed the transition from early spermatid stage.ConclusionWe characterize a new function for elav-like genes, showing that melav2 plays a crucial role during spermatid differentiation, especially in the regulation of chromatin condensation and/or cell elongation.

Correlation Between CFTR Gene Mutations in Iranian Men With Congenital Absence of the Vas Deferens and Anatomical Genital Phenotype

Congenital bilateral absence of the vas deferens (CBAVD) and congenital unilateral absence of the vas deferens (CUAVD) are 2 causes of male sterility; these phenotypes are found in 1%-2% of men investigated for infertility and approximately 10% of men with azoospermia. To study the correlation between genital phenotype and cystic fibrosis genotype in men lacking at least 1 vas deferens, we evaluated the role of different CFTR gene mutations in the morphologic genital phenotype of 119 infertile men with bilateral or unilateral absence of the vas deferens (112 CBAVD and 7 CUAVD patients). Renal, scrotal, and transrectal ultrasonography were systematically performed. CFTR mutations and (TG)m(T)n polymorphism were analyzed, and epididymal and seminal vesicular abnormalities and testicular volume were compared among men with 2, 1, or no CFTR gene mutation, with or without the 5T allele. Our results showed that patients with CBAVD and renal agenesis have the same reproductive tract abnormalities as those with CUAVD, and reproductive tract abnormalities were independent of the subtypes of CFTR genotype in patients with absence of the vas deferens and CFTR gene mutations. Seminal vesicles did not differ between patients with or without CFTR gene mutation, but epididymal abnormalities were more frequent in CBAVD men without the mutation. Low testicular volume was observed in CBAVD men without the CFTR and IVS8-5T mutations, so we can hypothesize that a testicular factor (genetic or environmental) rather than CFTR gene mutations plays a role in determining the phenotype. Further studies using common diagnostic criteria are required to confirm our observations.

FNDC3A is required for adhesion between spermatids and Sertoli cells

Symplastic spermatids (sys) male mice are sterile due to a recessive mutation that causes defective adhesion between spermatids and Sertoli cells within the seminiferous epithelium. We show that the mutation in sys mice involves a deletion of 1.24 Mb of chromosome 14. Comparative genomic analysis suggests that this region contains only one gene, Fndc3a. A genetic complementation analysis using mice with a specific mutation within Fndc3a verifies that mutation of Fndc3a is the cause of male sterility in sys mice. Fndc3a is a member of a three-gene family in mice. Fndc3a, which is expressed in several tissues including testis, encodes a novel protein composed of a proline-rich amino-terminus, nine fibronectin type-III domains, and a hydrophobic carboxy-terminus. The proline-rich region of each family member contains conserved amino acids that include a PPGY consensus binding site for type I WW domain containing proteins. The hydrophobic carboxy-terminus is similar to that found in ‘tail-anchored’ proteins, integral membrane proteins that are localized to the cytosolic face of the endoplasmic reticulum. Immunohistochemical staining indicated that FNDC3A localizes to the acrosome of spermatids, as well as to Leydig cells in the mouse testis. Acrosomal localization of FNDC3A is observed in spermatids between step 2 and step 10 inclusive. In step 12 spermatids, FNDC3A is largely absent from the acrosomal region with immunostaining being localized to vesicular structures located within the cytoplasm of elongate spermatids. Models are presented for the function of FNDC3A in mediating spermatid–Sertoli adhesion during mouse spermatogenesis.

A male-sterile mutation in soybean (Glycine max) affecting chromosome arrangement in metaphase plate and cytokinesis

A spontaneous male-sterile, female-fertile mutation affecting bivalent arrangement at the metaphase plate and cytokinesis was detected in line BR98-197 of the soybean breeding program developed by Embrapa - National Soybean Research Centre. Untill diakinesis, meiosis was normal with chromosome pairing as bivalents. From this phase, in several meiocytes, bivalents were not able to organize a single metaphase plate and remained scattered in the cytoplasm in a few or several groups. In these meiocytes, chromosomes segregated in both divisions giving rise to several micronuclei. However, the main cause of male sterility was the absence of cytokinesis after telophase II. Instead of the typical tetrads of microspores, four nucleate coenocytic microspores were formed. In the mutant, pollen mitoses did not occur, and after engorgement by starch, pollen underwent a progressive process of degeneration.

Deletion of the Parkin coregulated gene causes male sterility in the quaking(viable) mouse mutant.

Quaking(viable) (qk(v)) is a recessive neurological mouse mutation with severe dysmyelination of the CNS and spermiogenesis failure. The molecular lesion in the qk(v) mutant is a deletion of approximately 1 Mb on mouse chromosome 17 that alters the expression of the qk gene in oligodendrocytes. Complementation analysis between the qk(v) mutation and qk mutant alleles generated through chemical mutagenesis showed that the male sterility is a distinctive feature of the qk(v) allele. This observation suggested that the sperm differentiation defect in qk(v) is due to the deletion of a gene(s) distinct from qk. Here, we demonstrate that the deletion of Pacrg is the cause of male sterility in the qk(v) mutant. Pacrg is the mouse homologue of the human PARKIN-coregulated gene (PACRG), which encodes for a protein whose biochemical function remains unclear. We show that Pacrg is highly expressed in the testes in both mice and humans. In addition, the expression pattern of Pacrg during spermiogenesis suggests that it plays a role in sperm differentiation. In support of this hypothesis, we show that transgenic expression of Pacrg in testes restores spermiogenesis and fertility in qk(v) males. This finding provides the first in vivo evidence, to our knowledge, for the function of Pacrg in a model organism. Immunolocalization experiments on isolated spermatozoa show that the Pacrg protein is present in mature sperm. Remarkably, the mammalian Pacrg protein shares significant sequence similarities with gene products from flagellated protozoans, suggesting that Pacrg may be necessary for proper flagellar formation in many organisms.

Cytogenetic Analysis of Thermosensitive Genic Male Sterility (TGMS) Recovered from a Pennisetum glaucum (L.) R. Br.×P. violaceum (Lam.) L. Rich Cross

One male-sterile plant was recovered from a cross between pearl millet (Pennisetum glaucum) and its wild progenitor P. violaceum. The behavior of male sterility was temperature dependent, as the development of pollen mother cells to pollen grains was found to be dependent on temperature. The plant showed sterility at lower temperatures while it was fertile at higher temperatures during seasonal variations. This thermosensitive male sterility trait was found to be genetically controlled based on the segregation data of backcross generation. Segregation was observed for ca. 50% of the population expressing this trait. These TGMS plants exhibited transition from fertility to sterility when mean daily temperature reduced to ca. 20°C or below, and fertility was restored above this temperature. The period of 4 to 5 d prior to anthesis was found to be most sensitive to temperature variations. Chromosomal studies on the parent plant as well as segregants showed 2n=14 with regular meiosis during non-sensitive phase (period exhibiting male-fertility) ruling out aneuploidy and/or gross chromosomal structural aberrations as a cause of male sterility. Fertility characters were also studied for TGMS and non-TGMS plants during sensitive and non-sensitive phases. Pollen grains shed from the TGMS plants were much more vulnerable than from normal plants during sensitive phase indicating poor development of the membrane and walls of the pollen grains during sterility inducing conditions. Transition characteristics from male sterility to male fertility during sensitive and non-sensitive phases for TGMS and non-TGMS plants are discussed.

Further cytological characteristics of a male‐sterile mutant in soybean affecting cytokinesis and microspore development

AbstractA spontaneous male‐sterile mutation affecting microspore development has been found in BR97‐13509H line of the official Brazilian soybean breeding programme. Slightly abnormal and irregular chromosome segregation occurred in both meiotic divisions, albeit insufficient to explain total pollen sterility. The main cause of male sterility was the absence of cytokinesis after telophase II. Instead of the typical tetrads of microspores, four‐nucleate coenocytic microspores were formed. Coenocytic microspores resulting from the absence of cytokinesis have also been described in ms1 and ms4 male‐sterile soybean mutants, although with certain differential characteristics. In the BR97‐13509H mutant line, after interphase the four nuclei were rejoined in one or two metaphase plates and progressed through the first pollen mitosis. Pollen then underwent a progressive process of degeneration characterized by continuous cytoplasmic shrinkage. The second pollen mitosis failed to occur, and pollen sterility became total.

The "drought-inducible" histone H1s of tobacco play no role in male sterility linked to alterations in H1 variants.

Tobacco ( Nicotiana tabacum L.) has two major H1 variants (H1A and H1B), which account for over 80% of chromatin linker histones, and four minor variants: H1C, H1D, H1E and H1F. We have shown previously [M. Prymakowska-Bosak et al. (1999) Plant Cell 11:2317-2329] that reversal of the natural proportion of major to minor H1 variants in transgenic tobacco plants results in a characteristic male-sterility phenotype identical to that occurring in many plant species subjected to water deficit at the time of male meiosis. It has been proposed by others that the drought-induced arrest of male gametophyte development is linked to decreased sugar delivery to reproductive tissues. Within the family of angiosperm H1s there is a well-defined class of minor H1 variants named "drought inducible" because some of its members have been shown to be induced by water deficit. We have identified and cloned the tobacco H1C gene, which, based on sequence similarity, represents a "drought-inducible" minor H1 variant. Analysis of the un-translated mRNA and promoter regions of H1C suggests a regulation by sucrose concentration. Antisense silencing of H1C and its close homologue H1D in plants that do not express H1A and H1B does not affect the characteristic H1A(-)/ H1B(-) male-sterility phenotype. Silencing of H1C and H1D also has no effect on growth and development of plants. Our findings demonstrate that H1C and H1D are dispensable for normal growth and development of tobacco, and that the compensatory up-regulation of "drought-inducible" H1s observed in H1A(-)/ H1B(-) plants is not the direct cause of male sterility linked to alterations in H1 variants.