Embryological observations on seed abortion in Hibiscus syriacus L. and physiological studies on nutrients, enzyme activity and endogenous hormones.

Under natural conditions, most Hibiscus syriacus L. individuals form very few mature seeds or the mature seeds that do form are of poor quality. As a result, seed yield is poor and seeds have low natural germinability. These phenomena strongly hinder utilization of the excellent germplasm resources of H. syriacus. The study has shown that pollen activity and stigma receptivity were high on the day of anthesis, and the pistils and stamens were fertile. Pollen release and stigma receptivity were synchronous. But in styles following self and cross-pollination, pollen tube abnormalities (distortion and twisting of the pollen tubes) and callose deposition were observed. Cross-pollinated pollen tubes elongated faster and fewer pollen tube abnormalities were observed compared with self-pollinated pollen tubes. And during embryo development, abnormalities during the heart-shaped embryo stage led to embryo abortion. Imbalance in antioxidant enzyme activities and low contents of auxin and cytokinin during early stages of embryo development may affect embryo development. Therefore, a low frequency of outcrossing and mid-development embryo abortion may be important developmental causes of H. syriacus seed abortion. Nutrient deficiencies, imbalance in antioxidant enzyme activities, and a high content of abscisic acid at advanced stages of seed development may be physiological causes of seed abortion.

Aridity weakens population-level effects of multiple species interactions on Hibiscus meyeri.

Predicting how species' abundances and ranges will shift in response to climate change requires a mechanistic understanding of how multiple factors interact to limit population growth. Both abiotic stress and species interactions can limit populations and potentially set range boundaries, but we have a poor understanding of when and where each is most critical. A commonly cited hypothesis, first proposed by Darwin, posits that abiotic factors (e.g., temperature, precipitation) are stronger determinants of range boundaries in apparently abiotically stressful areas ("stress" indicates abiotic factors that reduce population growth), including desert, polar, or high-elevation environments, whereas species interactions (e.g., herbivory, competition) play a stronger role in apparently less stressful environments. We tested a core tenet of this hypothesis-that population growth rate is more strongly affected by species interactions in less stressful areas-using experimental manipulations of species interactions affecting a common herbaceous plant, Hibiscus meyeri (Malvaceae), across an aridity gradient in a semiarid African savanna. Population growth was more strongly affected by four distinct species interactions (competition with herbaceous and shrubby neighbors, herbivory, and pollination) in less stressful mesic areas than in more stressful arid sites. However, contrary to common assumptions, this effect did not arise because of greater density or diversity of interacting species in less stressful areas, but rather because aridity reduced sensitivity of population growth to these interactions. Our work supports classic predictions about the relative strength of factors regulating population growth across stress gradients, but suggests that this pattern results from a previously unappreciated mechanism that may apply to many species worldwide.

Reactive Oxygen Species Accumulation Strongly Allied with Genetic Male Sterility Convertible to Cytoplasmic Male Sterility in Kenaf.

Male sterility (MS) plays a key role in the hybrid breed production of plants. Researchers have focused on the association between genetic male sterility (GMS) and cytoplasmic male sterility (CMS) in kenaf. In this study, P9BS (a natural GMS mutant of the kenaf line P9B) and male plants of P9B were used as parents in multiple backcross generations to produce P9SA, a CMS line with stable sterility, to explore the molecular mechanisms of the association between GMS and CMS. The anthers of the maintainer (P9B), GMS (P9BS), and CMS (P9SA) lines were compared through phenotypic, cell morphological, physiological, biochemical observations, and transcriptome analysis. Premature degradation of the tapetum was observed at the mononuclear stage in P9BS and P9SA, which also had lower activity of reactive oxygen species (ROS) scavenging enzymes compared with P9B. Many coexpressed differentially expressed genes were related to ROS balance, including ATP synthase, electron chain transfer, and ROS scavenging processes were upregulated in P9B. CMS plants had a higher ROS accumulation than GMS plants. The MDA content in P9SA was 3.2 times that of P9BS, and therefore, a higher degree of abortion occurred in P9SA, which may indicate that the conversion between CMS and GMS is related to intracellular ROS accumulation. Our study adds new insights into the natural transformation of GMS and CMS in plants in general and kenaf in particular.

Comparative acetylomic analysis reveals differentially acetylated proteins regulating anther and pollen development in kenaf cytoplasmic male sterility line.

Cytoplasmic male sterility (CMS) is widely used in plant breeding and represents a perfect model to understand cyto-nuclear interactions and pollen development research. Lysine acetylation in proteins is a dynamic and reversible posttranslational modification (PTM) that plays an important roles in diverse cell processes and signaling. However, studies addressing acetylation PTM regarding to anther and pollen development in CMS background are largely lacking. To reveal the possible mechanism of kenaf (Hibiscus cannabinus L.) CMS and pollen development, we performed a label-free-based comparative acetylome analysis in kenaf anther of a CMS line and wild-type (Wt). Using whole transcriptome unigenes of kenaf as the reference genome, we identified a total of 1204 Kac (lysin acetylation) sites on 1110 peptides corresponding to 672 unique proteins. Futher analysis showed 56 out of 672 proteins were differentially acetylated between CMS and Wt line, with 13 and 43 of those characterized up- and downregulated, respectively. Thirty-eight and 82 proteins were detected distinctively acetylated in CMS and Wt lines, respectively. And evaluation of the acetylomic and proteomic results indicated that the most significantly acetylated proteins were not associated with abundant changes at the protein level. Bioinformatics analysis demonstrated that many of these proteins were involved in various biological processes which may play key roles in pollen development, inculding tricarboxylic acid (TCA) cycle and energy metabolism, protein folding, protein metabolism, cell signaling, gene expression regulation. Taken together, our results provide insight into the CMS molecular mechanism and pollen development in kenaf from a protein acetylation perspective.

Molecular cloning and subcellular localization of six HDACs and their roles in response to salt and drought stress in kenaf (Hibiscus cannabinus L.).

BACKGROUND: Histone acetylation is an important epigenetic modification that regulates gene activity in response to stress. Histone acetylation levels are reversibly regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs). The imperative roles of HDACs in gene transcription, transcriptional regulation, growth and responses to stressful environment have been widely investigated in Arabidopsis. However, data regarding HDACs in kenaf crop has not been disclosed yet. RESULTS: In this study, six HDACs genes (HcHDA2, HcHDA6, HcHDA8, HcHDA9, HcHDA19, and HcSRT2) were isolated and characterized. Phylogenetic tree revealed that these HcHDACs shared high degree of sequence homology with those of Gossypium arboreum. Subcellular localization analysis showed that GFP-tagged HcHDA2 and HcHDA8 were predominantly localized in the nucleus, HcHDA6 and HcHDA19 in nucleus and cytosol. The HcHDA9 was found in both nucleus and plasma membranes. Real-time quantitative PCR showed that the six HcHDACs genes were expressed with distinct expression patterns across plant tissues. Furthermore, we determined differential accumulation of HcHDACs transcripts under salt and drought treatments, indicating that these enzymes may participate in the biological process under stress in kenaf. Finally, we showed that the levels of histone H3 and H4 acetylation were modulated by salt and drought stress in kenaf. CONCLUSIONS: We have isolated and characterized six HDACs genes from kenaf. These data showed that HDACs are imperative players for growth and development as well abiotic stress responses in kenaf.

RNA-seq for comparative transcript profiling of kenaf under salinity stress.

Kenaf (Hibiscus cannabinus L.) is an economically important global natural fiber crop. As a consequence of the increased demand for food crops and the reduction of available arable land, kenaf cultivation has increasingly shifted to saline and alkaline land. To investigate the molecular mechanism of salinity tolerance in kenaf, we performed Illumina high-throughput RNA sequencing on shoot tips of kenaf and identified 71,318 unigenes, which were annotated using four different protein databases. In total, 2,384 differentially expressed genes (DEGs) were identified between the salt-stressed and the control plants, 1,702 of these transcripts were up-regulated and 683 transcripts were down-regulated. Thirty-seven transcripts belonging to 15 transcription-factor families that respond to salt stress were identified. Gene ontology function enrichment analysis revealed that the genes encoding antioxidant enzymes were up-regulated. The amino acid metabolism and carbohydrate metabolism pathways were highly enriched among these DEGs under salt stress conditions. In order to confirm the RNA-seq data, we randomly selected 20 unigenes for analysis using a quntitative real-time polymerase chain reaction. Our study not only provided the large-scale assessment of transcriptome resources of kenaf but also guidelines for understanding the mechanism underlying salt stress responses in kenaf.

Physico-chemical changes in karkade (Hibiscus sabdariffa L.) seedlings responding to salt stress.

Salinity is one of the major abiotic stress factors affecting series of morphological, physiological, metabolic and molecular changes in plant growth. The effect of different concentrations (0, 25, 50, 100 and 150 mM) of NaCl on the vegetative growth and some physiological parameters of karkade (Hibiscus sabdariffa var. sabdariffa) seedling were investigated. NaCl affected the germination rate, delayed emergence and retarded vegetative growth of seedlings. The length of seedling as well as the leaf area was significantly reduced. The fresh weight remained lower in NaCl treated seedlings compared to control. NaCl at 100 and 150 mM concentrations had significant effect on the dry matter contents of the treated seedlings. The chloroplast pigments in the treated seedlings were affected, suggesting that the NaCl had a significant effect on the chlorophyll and carotenoid biosynthesis. The results showed that the salt treatments induced an increase in proline concentration of the seedlings. The osmotic potential (ψs) of NaCl treated seedlings decreased with increasing NaCl concentrations. Salt treatments resulted in dramatic quantitative reduction in the total sterol percent compared with control ones. Salt stress resulted in increase and decrease of Na+ and K+ ions, respectively. NaCl salinity increased lipid peroxidation. SDS-PAGE was used to evaluate protein pattern after applying salt stress. High molecular weight proteins were intensified, while low molecular weight proteins were faint. NaCl at 100 and 150 mM concentration distinguished with new protein bands. Salt stress induced a new peroxidase bands and increased the band intensity, indicating the protective role of peroxidase enzyme.

Rapid modulation of ultraviolet shielding in plants is influenced by solar ultraviolet radiation and linked to alterations in flavonoids.

The accumulation of ultraviolet (UV)-absorbing compounds (flavonoids and related phenylpropanoids) and the resultant decrease in epidermal UV transmittance (TUV ) are primary protective mechanisms employed by plants against potentially damaging solar UV radiation and are critical components of the overall acclimation response of plants to changing solar UV environments. Whether plants can adjust this UV sunscreen protection in response to rapid changes in UV, as occurs on a diurnal basis, is largely unexplored. Here, we use a combination of approaches to demonstrate that plants can modulate their UV-screening properties within minutes to hours, and these changes are driven, in part, by UV radiation. For the cultivated species Abelmoschus esculentus, large (30-50%) and reversible changes in TUV occurred on a diurnal basis, and these adjustments were associated with changes in the concentrations of whole-leaf UV-absorbing compounds and several quercetin glycosides. Similar results were found for two other species (Vicia faba and Solanum lycopersicum), but no such changes were detected in Zea mays. These findings reveal a much more dynamic UV-protection mechanism than previously recognized, raise important questions concerning the costs and benefits of UV-protection strategies in plants and have practical implications for employing UV to enhance crop vigor and quality in controlled environments.

Spatial and temporal transcriptome changes occurring during flower opening and senescence of the ephemeral hibiscus flower, Hibiscus rosa-sinensis.

Flowers are complex systems whose vegetative and sexual structures initiate and die in a synchronous manner. The rapidity of this process varies widely in flowers, with some lasting for months while others such as Hibiscus rosa-sinensis survive for only a day. The genetic regulation underlying these differences is unclear. To identify key genes and pathways that coordinate floral organ senescence of ephemeral flowers, we identified transcripts in H. rosa-sinensis floral organs by 454 sequencing. During development, 2053 transcripts increased and 2135 decreased significantly in abundance. The senescence of the flower was associated with increased abundance of many hydrolytic genes, including aspartic and cysteine proteases, vacuolar processing enzymes, and nucleases. Pathway analysis suggested that transcripts altering significantly in abundance were enriched in functions related to cell wall-, aquaporin-, light/circadian clock-, autophagy-, and calcium-related genes. Finding enrichment in light/circadian clock-related genes fits well with the observation that hibiscus floral development is highly synchronized with light and the hypothesis that ageing/senescence of the flower is orchestrated by a molecular clock. Further study of these genes will provide novel insight into how the molecular clock is able to regulate the timing of programmed cell death in tissues.

The flower of Hibiscus trionum is both visibly and measurably iridescent.

Living organisms can use minute structures to manipulate the reflection of light and display colours based on interference. There has been debate in recent literature over whether the diffractive optical effects produced by epoxy replicas of petals with folded cuticles persist and induce iridescence in the original flowers when the effects of petal pigment and illumination are taken into account. We explored the optical properties of the petal of Hibiscus trionum by macro-imaging, scanning and transmission electron microscopy, and visible and ultraviolet (UV) angle-resolved spectroscopy of the petal. The flower of Hibiscus trionum is visibly iridescent, and the iridescence can be captured photographically. The iridescence derives from a diffraction grating generated by folds of the cuticle. The iridescence of the petal can be quantitatively characterized by spectrometric measurements with several square-millimetres of sample area illuminated. The flower of Hibiscus trionum has the potential to interact with its pollinators (honeybees, other bees, butterflies and flies) through iridescent signals produced by its cuticular diffraction grating.

Iridescent flowers? Contribution of surface structures to optical signaling.

The color of natural objects depends on how they are structured and pigmented. In flowers, both the surface structure of the petals and the pigments they contain determine coloration. The aim of the present study was to assess the contribution of structural coloration, including iridescence, to overall floral coloration. We studied the reflection characteristics of flower petals of various plant species with an imaging scatterometer, which allows direct visualization of the angle dependence of the reflected light in the hemisphere above the petal. To separate the light reflected by the flower surface from the light backscattered by the components inside (e.g. the vacuoles), we also investigated surface casts. A survey among angiosperms revealed three different types of floral surface structure, each with distinct reflections. Petals with a smooth and very flat surface had mirror-like reflections and petal surfaces with cones yielded diffuse reflections. Petals with striations yielded diffraction patterns when single cells were illuminated. The iridescent signal, however, vanished when illumination similar to that found in natural conditions was applied. Pigmentary rather than structural coloration determines the optical appearance of flowers. Therefore, the hypothesized signaling by flowers with striated surfaces to attract potential pollinators presently seems untenable.

Determination of suitable microspore stage and callus induction from anthers of kenaf (Hibiscus cannabinus L.).

Kenaf (Hibiscus cannabinus L.) is one of the important species of Hibiscus cultivated for fiber. Availability of homozygous parent lines is prerequisite to the use of the heterosis effect reproducible in hybrid breeding. The production of haploid plants by anther culture followed by chromosome doubling can be achieved in short period compared with inbred lines by conventional method that requires self pollination of parent material. In this research, the effects of the microspore developmental stage, time of flower collection, various pretreatments, different combinations of hormones, and culture condition on anther culture of KB6 variety of Kenaf were studied. Young flower buds with immature anthers at the appropriate stage of microspore development were sterilized and the anthers were carefully dissected from the flower buds and subjected to various pretreatments and different combinations of hormones like NAA, 2,4-D, Kinetin, BAP, and TDZ to induce callus. The best microspore development stage of the flower buds was about 6-8 mm long collected 1-2 weeks after flower initiation. At that stage, the microspores were at the uninucleate stage which was suitable for culture. The best callus induction frequency was 90% in the optimized semisolid MS medium fortified with 3.0 mg/L BAP + 3.0 mg/L NAA.

A cyclic nucleotide-gated channel gene HcCNGC21 positively regulates salt and drought stress responses in kenaf (Hibiscus cannabinus L.).

Cyclic Nucleotide-Gated Channels (CNGCs) serve as Ca2+ permeable cation transport pathways, which are involved in the regulation of various biological functions such as plant cell ion selective permeability, growth and development, responses to biotic and abiotic stresses. At the present study, a total of 31 CNGC genes were identified and bioinformatically analyzed in kenaf. Among these genes, HcCNGC21 characterized to localize at the plasma membrane, with the highest expression levels in leaves, followed by roots. In addition, HcCNGC21 could be significantly induced under salt or drought stress. Virus-induced gene silencing (VIGS) of HcCNGC21 in kenaf caused notable growth inhibition under salt or drought stress, characterized by reductions in plant height, stem diameter, leaf area, root length, root surface area, and root tip number. Meanwhile, the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) were significantly decreased, accompanied by reduced levels of osmoregulatory substances and total chlorophyll content. However, ROS accumulation and Na+ content increased. The expression of stress-responsive genes, such as HcSOD, HcPOD, HcCAT, HcERF3, HcNAC29, HcP5CS, HcLTP, and HcNCED, was significantly downregulated in these silenced lines. However, under salt or drought stress, the physiological performance and expression of stress-related genes in transgenic Arabidopsis thaliana plants overexpressing HcCNGC21 were diametrically opposite to those of TRV2-HcCNGC21 kenaf line. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays revealed that HcCNGC21 interacts with HcAnnexin D1. These findings collectively underscore the positive role of HcCNGC21 in plant resistance to salt and drought stress.

Leaf photosynthesis and respiration of three bioenergy crops in relation to temperature and leaf nitrogen: how conserved are biochemical model parameters among crop species?

Given the need for parallel increases in food and energy production from crops in the context of global change, crop simulation models and data sets to feed these models with photosynthesis and respiration parameters are increasingly important. This study provides information on photosynthesis and respiration for three energy crops (sunflower, kenaf, and cynara), reviews relevant information for five other crops (wheat, barley, cotton, tobacco, and grape), and assesses how conserved photosynthesis parameters are among crops. Using large data sets and optimization techniques, the C(3) leaf photosynthesis model of Farquhar, von Caemmerer, and Berry (FvCB) and an empirical night respiration model for tested energy crops accounting for effects of temperature and leaf nitrogen were parameterized. Instead of the common approach of using information on net photosynthesis response to CO(2) at the stomatal cavity (A(n)-C(i)), the model was parameterized by analysing the photosynthesis response to incident light intensity (A(n)-I(inc)). Convincing evidence is provided that the maximum Rubisco carboxylation rate or the maximum electron transport rate was very similar whether derived from A(n)-C(i) or from A(n)-I(inc) data sets. Parameters characterizing Rubisco limitation, electron transport limitation, the degree to which light inhibits leaf respiration, night respiration, and the minimum leaf nitrogen required for photosynthesis were then determined. Model predictions were validated against independent sets. Only a few FvCB parameters were conserved among crop species, thus species-specific FvCB model parameters are needed for crop modelling. Therefore, information from readily available but underexplored A(n)-I(inc) data should be re-analysed, thereby expanding the potential of combining classical photosynthetic data and the biochemical model.

Cuticle chemistry drives the development of diffraction gratings on the surface of Hibiscus trionum petals.

Plants combine both chemical and structural means to appear colorful. We now have an extensive understanding of the metabolic pathways used by flowering plants to synthesize pigments, but the mechanisms remain obscure whereby cells produce microscopic structures sufficiently regular to interfere with light and create an optical effect. Here, we combine transgenic approaches in a novel model system, Hibiscus trionum, with chemical analyses of the cuticle, both in transgenic lines and in different species of Hibiscus, to investigate the formation of a semi-ordered diffraction grating on the petal surface. We show that regulating both cuticle production and epidermal cell growth is insufficient to determine the type of cuticular pattern produced. Instead, the chemical composition of the cuticle plays a crucial role in restricting the formation of diffraction gratings to the pigmented region of the petal. This suggests that buckling, driven by spatiotemporal regulation of cuticle chemistry, could pattern the petal surface at the nanoscale.

Cytotype Affects the Capability of the Whitefly Bemisia tabaci MED Species To Feed and Oviposit on an Unfavorable Host Plant.

The acquisition of nutritional obligate primary endosymbionts (P-symbionts) allowed phloemo-phageous insects to feed on plant sap and thus colonize novel ecological niches. P-symbionts often coexist with facultative secondary endosymbionts (S-symbionts), which may also influence their hosts' niche utilization ability. The whitefly Bemisia tabaci is a highly diversified species complex harboring, in addition to the P-symbiont "Candidatus Portiera aleyrodidarum," seven S-symbionts whose roles remain poorly understood. Here, we compare the phenotypic and metabolic responses of three B. tabaci lines differing in their S-symbiont community, reared on three different host plants, hibiscus, tobacco, or lantana, and address whether and how S-symbionts influence insect capacity to feed and produce offspring on those plants. We first show that hibiscus, tobacco, and lantana differ in their free amino acid composition. Insects' performance, as well as free amino acid profile and symbiotic load, were shown to be plant dependent, suggesting a critical role for the plant nutritional properties. Insect fecundity was significantly lower on lantana, indicating that it is the least favorable plant. Remarkably, insects reared on this plant show a specific amino acid profile and a higher symbiont density compared to the two other plants. In addition, this plant was the only one for which fecundity differences were observed between lines. Using genetically homogeneous hybrids, we demonstrate that cytotype (mitochondria and symbionts), and not genotype, is a major determinant of females' fecundity and amino acid profile on lantana. As cytotypes differ in their S-symbiont community, we propose that these symbionts may mediate their hosts' suitable plant range. IMPORTANCE Microbial symbionts are universal in eukaryotes, and it is now recognized that symbiotic associations represent major evolutionary driving forces. However, the extent to which symbionts contribute to their hosts' ecological adaptation and subsequent diversification is far from being fully elucidated. The whitefly Bemisia tabaci is a sap feeder associated with multiple coinfecting intracellular facultative symbionts. Here, we show that plant species simultaneously affect whiteflies' performance, amino acid profile, and symbiotic density, which could be partially explained by differences in plant nutritional properties. We also demonstrate that, on lantana, the least favorable plant used in our study, whiteflies' performance is determined by their cytotype. We propose that the host plant utilization in B. tabaci is influenced by its facultative symbiont community composition, possibly through its impact on the host dietary requirements. Altogether, our data provide new insights into the impact of intracellular microorganisms on their animal hosts' ecological niche range and diversification.

Distribution, epidemiology and molecular variability of the begomovirus complexes associated with yellow vein mosaic disease of mesta in India.

Yellow vein mosaic disease of mesta (Hibiscus spp.) poses a serious threat to the cultivation of this crop in India. The disease was found to be associated with two different whitefly-transmitted monopartite begomoviruses, Mesta yellow vein mosaic virus and Mesta yellow vein mosaic Bahraich virus, together with two betasatellite species, Cotton leaf curl Multan betasatellite and Ludwigia leaf distortion betasatellite. These begomovirus complexes were detected in different combinations throughout the mesta growing regions of India. All the eight cultivars tested were highly susceptible to the disease. The effect of the disease in terms of loss in fibre yield was greatest (around 70%) in plants that were inoculated at an early stage of growth. A regression approach was adopted to consider the relationship of whitefly vector populations with weather conditions and disease spread which explained that different conducive weather factors facilitated the build up of whitefly populations and contributed to the spread of the disease.

[pH-dependent regulation of electron transport in chloroplasts. Computer simulation investigation].

A mathematical model for the description of the key stages of photosynthetic electron transport and transmembrane proton transfer in chloroplasts is presented. Numerical modeling of electron and proton transport with due regard for regulatory processes on the donor and acceptor sites of photosystem I (PS I) was performed. The influence of pH-dependent activation of the Calvin cycle enzymes and energy dissipation in PS II (nonphotochemical quenching of chlorophyll fluorescence) on the kinetics of light-reduced redox transients of P700, plastoquinone and NADPH, as well as intrathylakoid pH(in), and ATP was studied. It was demonstrated that pH-dependent regulatory processes influence the distribution of electron fluxes on the acceptor site of PS I and the total rate of electron flow between PS II and PS I. The light-induced activation of the Calvin cycle enzymes leads to a significant acceleration of the electron flow to NADP+ and the reduction of the electron flow from PS 1 to molecular oxygen.