Ectopic expression of SOD and APX genes in Arabidopsis alters metabolic pools and genes related to secondary cell wall cellulose biosynthesis and improve salt tolerance.

Hydrogen peroxide (H2O2) is known to accumulate in plants during abiotic stress conditions and also acts as a signalling molecule. In this study, Arabidopsis thaliana transgenics overexpressing cytosolic CuZn-superoxide dismutase (PaSOD) from poly-extremophile high-altitude Himalayan plant Potentilla atrosanguinea, cytosolic ascorbate peroxidase (RaAPX) from Rheum australe and dual transgenics overexpressing both the genes were developed and analyzed under salt stress. In comparison to wild-type (WT) or single transgenics, the performance of dual transgenics under salt stress was better with higher biomass accumulation and cellulose content. We identified genes involved in cell wall biosynthesis, including nine cellulose synthases (CesA), seven cellulose synthase-like proteins together with other wall-related genes. RNA-seq analysis and qPCR revealed differential regulation of genes (CesA 4, 7 and 8) and transcription factors (MYB46 and 83) involved in secondary cell wall cellulose biosynthesis, amongst which most of the cellulose biosynthesis gene showed upregulation in single (PaSOD line) and dual transgenics at 100 mM salt stress. A positive correlation between cellulose content and H2O2 accumulation was observed in these transgenic lines. Further, cellulose content was 1.6-2 folds significantly higher in PaSOD and dual transgenic lines, 1.4 fold higher in RaAPX lines as compared to WT plants under stress conditions. Additionally, transgenics overexpressing PaSOD and RaAPX also displayed higher amounts of phenolics as compared to WT. The novelty of present study is that H2O2 apart from its role in signalling, it also provides mechanical strength to plants and aid in plant biomass production during salt stress by transcriptional activation of cellulose biosynthesis pathway. This modulation of the cellulose biosynthetic machinery in plants has the potential to provide insight into plant growth, morphogenesis and to create plants with enhanced cellulose content for biofuel use.

The TRAF Mediated Gametogenesis Progression (TRAMGaP) Gene Is Required for Megaspore Mother Cell Specification and Gametophyte Development.

In plants, the role of TRAF-like proteins with meprin and the TRAF homology (MATH) domain is far from clear. In animals, these proteins serve as adapter molecules to mediate signal transduction from Tumor Necrosis Factor Receptor to downstream effector molecules. A seed-sterile mutant with a disrupted TRAF-like gene (At5g26290) exhibiting aberrant gametogenesis led us to investigate the developmental role of this gene in Arabidopsis (Arabidopsis thaliana). The mutation was semidominant and resulted in pleiotropic phenotypes with such features as short siliques with fewer ovules, pollen and seed sterility, altered Megaspore Mother Cell (MMC) specification, and delayed programmed cell death in megaspores and the tapetum, features that overlapped those in other well-characterized mutants. Seed sterility and reduced transmission frequency of the mutant alleles pointed to a dual role, sporophytic and gametophytic, for the gene on the male side. The mutant also showed altered expression of various genes involved in such cellular and developmental pathways as regulation of transcription, biosynthesis and transport of lipids, hormone-mediated signaling, and gametophyte development. The diverse phenotypes of the mutant and the altered expression of key genes related to gametophyte and seed development could be explained based on the functional similarly between At5g26290 and MATH-BTB domain proteins that modulate gene expression through the ubiquitin-mediated proteasome system. These results show a novel link between a TRAF-like gene and reproductive development in plants.

Comprehensive Chemical Profiling of Picrorhiza kurroa Royle ex Benth Using NMR, HPTLC and LC-MS/MS Techniques.

Picrorhiza kurroa is an important herb in Indian medicine and contains cucurbitacins, flavonoids, phenolics, iridoid-glucoside and their derivatives as active constituents for the treatment of indigestion, fever, hepatitis, cancer, liver and respiratory diseases. Extensive use of P. kurroa needs detailed analysis and recognition of chemical diversity, is of great importance to evaluate their role as quality control markers. In the present study, comprehensive metabolic profiling of crude extracts of leaves and rhizomes of P. kurroa was carried out using NMR, HPTLC and LC-MS/MS. Primary and secondary metabolites were unambiguously identified along with a new report of monoterpenic glycoside (1-ß-D-glucopyranosyl)-8-hydroxy-3,7-dimethyl-oct-2(E),6(E)-dienoate) in P. Kurroa. Significant qualitative differences with respect to the secondary metabolites were noticed between the leaves and rhizomes tissues. Leaves contained more cucurbitacins and flavonoids while iridoids were present more in rhizomes. The comprehensive chemical profiling is expected to give an idea of chemical diversity and quality of P. kurroa, for their ultimate utilisation in various applications.

Biotechnological approaches to the production of shikonins: a critical review with recent updates.

Shikonins are commercially important secondary compounds, known for array of biological activities such as antimicrobial, insecticidal, antitumor, antioxidants, etc. These compounds are usually colored and therefore have application in food, textiles and cosmetics. Shikonin and its derivatives, which are commercially most important of the naphthoquinone pigments, are distributed among members of the family Boraginaceae. These include different species of Lithospermum, Arnebia, Alkanna, Anchusa, Echium and Onosma. The growing demand for plant-based natural products has made this group of compounds one of the enthralling targets for their in vitro production. The aim of this review is to highlight the recent progress in production of shikonins by various biotechnological means. Different methods of increasing the levels of shikonins in plant cells such as selection of cell lines, optimization of culture conditions, elicitation, in situ product removal, genetic transformation and metabolic engineering are discussed. The experience of different researchers working worldwide on this aspect is also considered. Further, to meet market demand, the needs for continuous and reliable production systems, as well as future prospects, are included.

Crop-ecology and nutritional variability influence growth and secondary metabolites of Stevia rebaudiana Bertoni.

BACKGROUND: Plant nutrition and climatic conditions play important roles on the growth and secondary metabolites of stevia (Stevia rebaudiana Bertoni); however, the nutritional dose is strongly governed by the soil properties and climatic conditions of the growing region. In northern India, the interactive effects of crop ecology and plant nutrition on yield and secondary metabolites of stevia are not yet properly understood. Thus, a field experiment comprising three levels of nitrogen, two levels of phosphorus and three levels of potassium was conducted at three locations to ascertain whether the spatial and nutritional variability would dominate the leaf yield and secondary metabolites profile of stevia. RESULTS: Principal component analysis (PCA) indicates that the applications of 90 kg N, 40 kg P2O5 and 40 kg K2O ha-1 are the best nutritional conditions in terms of dry leaf yield for CSIR-IHBT (Council of Scientific and Industrial Research- Institute Himalayan Bioresource Technology) and RHRS (Regional Horticultural Research Station) conditions. The spatial variability also exerted considerable effect on the leaf yield and stevioside content in leaves. Among the three locations, CSIR-IHBT was found most suitable in case of dry leaf yield and secondary metabolites accumulation in leaves. CONCLUSIONS: The results suggest that dry leaf yield and accumulation of stevioside are controlled by the environmental factors and agronomic management; however, the accumulation of rebaudioside-A (Reb-A) is not much influenced by these two factors. Thus, leaf yield and secondary metabolite profiles of stevia can be improved through the selection of appropriate growing locations and proper nutrient management.

Expression of SOD and APX genes positively regulates secondary cell wall biosynthesis and promotes plant growth and yield in Arabidopsis under salt stress.

Abiotic stresses cause accumulation of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2) in plants. Sophisticated mechanisms are required to maintain optimum level of H2O2 that acts as signalling molecule regulating adaptive response to salt stress. CuZn-superoxide dismutase (CuZn-SOD) and ascorbate peroxidase (APX) constitute first line of defence against oxidative stress. In the present study, PaSOD and RaAPX genes from Potentilla atrosanguinea and Rheum australe, respectively were overexpressed individually as well as in combination in Arabidopsis thaliana. Interestingly, PaSOD and dual transgenic lines exhibit enhanced lignin deposition in their vascular bundles with altered S:G ratio under salt stress. RNA-seq analysis revealed that expression of PaSOD gene in single and dual transgenics positively regulates expression of lignin biosynthesis genes and transcription factors (NACs, MYBs, C3Hs and WRKY), leading to enhanced and ectopic deposition of lignin in vascular tissues with larger xylem fibres and alters S:G ratio, as well. In addition, transgenic plants exhibit growth promotion, higher biomass production and increased yield under salt stress as compared to wild type plants. Our results suggest that in dual transgenics, ROS generated during salt stress gets converted into H2O2 by SOD and its optimum level was maintained by APX. This basal level of H2O2 acts as messenger for transcriptional activation of lignin biosynthesis in vascular tissue, which provides mechanical strength to plants. These findings reveal an important role of PaSOD and RaAPX in enhancing salt tolerance of transgenic Arabidopsis via increased accumulation of compatible solutes and by regulating lignin biosynthesis.

Improved callus induction, shoot regeneration, and salt stress tolerance in Arabidopsis overexpressing superoxide dismutase from Potentilla atrosanguinea.

Superoxide dismutase (SOD) catalyzes the dismutation of superoxide radicals (O2( ·-)) to molecular oxygen (O2) and hydrogen peroxide (H2O2). Previously, we have identified and characterized a thermo-tolerant copper-zinc superoxide dismutase from Potentilla atrosanguinea (PaSOD), which retains its activity in the presence of NaCl. In the present study, we show that cotyledonary explants of PaSOD overexpressing transgenic Arabidopsis thaliana exhibit early callus induction and high shoot regenerative capacity than wild-type (WT) explants. Growth kinetic studies showed that transgenic lines have 2.6-3.3-folds higher growth rate of calli compared to WT. Regeneration frequency of calli developed from transgenic cotyledons was found to be 1.5-2.5-folds higher than that of WT explants on Murashige and Skoog medium supplemented with different concentrations of naphthalene acetic acid (NAA) and 6-benzylaminopurine (BAP) within 2 weeks. A positive regulatory effect of PaSOD and H2O2 was observed on different stages of callusing and regeneration. However, this effect was more pronounced at the early stages of the regeneration processes in transgenic lines as compared to WT. These results clearly indicate that plant regeneration is regulated by endogenous H2O2 and by factors, which enhance its accumulation. Transgenics also exhibited salt stress tolerance with higher SOD activity, chlorophyll content, total soluble sugars, and proline content, while lower ion leakage and less reduction in relative water content, as compared to WT. Thus, it appears that the activation of PaSOD at regeneration stage accompanied by increased H2O2 production can be one of the mechanisms controlling in vitro morphogenesis.

Genetic diversity of high-elevation populations of an endangered medicinal plant.

Intraspecific genetic variation in natural populations governs their potential to overcome challenging ecological and environmental conditions. In addition, knowledge of this variation is critical for the conservation and management of endangered plant taxa. Found in the Himalayas, Podophyllum hexandrum is an endangered high-elevation plant species that has great medicinal importance. Here we report on the genetic diversity analysis of 24 P. hexandrum populations (209 individuals), representing the whole of the Indian Himalayas. In the present study, seven amplified fragment length polymorphism (AFLP) primer pairs generated 1677 fragments, of which 866 were found to be polymorphic. Neighbour joining clustering, principal coordinate analysis and STRUCTURE analysis clustered 209 individuals from 24 populations of the Indian Himalayan mountains into two major groups with a significant amount of gene flow (Nm = 2.13) and moderate genetic differentiation Fst(0.196), G'st(0.20). This suggests that, regardless of geographical location, all of the populations from the Indian Himalayas are intermixed and are composed broadly of two types of genetic populations. High variance partitioned within populations (80 %) suggests that most of the diversity is restricted to the within-population level. These results suggest two possibilities about the ancient population structure of P. hexandrum: either all of the populations in the geographical region of the Indian Himalayas are remnants of a once-widespread ancient population, or they originated from two types of genetic populations, which coexisted a long time ago, but subsequently separated as a result of long-distance dispersal and natural selection. High variance partitioned within the populations indicates that these populations have evolved in response to their respective environments over time, but low levels of heterozygosity suggest the presence of historical population bottlenecks.

Simultaneous over-expression of PaSOD and RaAPX in transgenic Arabidopsis thaliana confers cold stress tolerance through increase in vascular lignifications.

Antioxidant enzymes play a significant role in eliminating toxic levels of reactive oxygen species (ROS), generated during stress from living cells. In the present study, two different antioxidant enzymes namely copper-zinc superoxide dismutase derived from Potentilla astrisanguinea (PaSOD) and ascorbate peroxidase (RaAPX) from Rheum austral both of which are high altitude cold niche area plants of Himalaya were cloned and simultaneously over-expressed in Arabidopsis thaliana to alleviate cold stress. It was found that the transgenic plants over-expressing both the genes were more tolerant to cold stress than either of the single gene expressing transgenic plants during growth and development. In both single (PaSOD, RaAPX) and double (PaSOD + RaAPX) transgenic plants higher levels of total antioxidant enzyme activities, chlorophyll content, total soluble sugars, proline content and lower levels of ROS, ion leakage were recorded when compared to the WT during cold stress (4°C), besides increase in yield. In the present study, Confocal and SEM analysis in conjunction with qPCR data on the expression pattern of lignin biosynthetic pathway genes revealed that the cold stress tolerance of the transgenic plants might be because of the peroxide induced up-regulation of lignin by antioxidant genes mediated triggering.

A simple technique for tracking individual spore and gametophyte development in Adiantum lunulatum Burm. f. using modified extra thin alginate film technique.

A new technique was developed for accurate calculation of percent germination and tracking of individual spores from germination to gametophyte development in Adiantum lunulatum. High percentage of ETAF immobilized spore germination (72.4%) was followed by development of gametophytic clumps. The ETAF immobilized clumps were cut into pieces and multiplied en masse. Apomictic sporophytes developed from the gametophytes. This indicated the potential of ETAF for mass propagation of A. lunulatum without the need to start from spores. Since individual spores can be tracked from germination to gametophyte development, the ETAF technique has the potential to be used for (i) harvesting uniformly developed plants of similar age for extensive experimentations and commercial utilization and (ii) detailed study on developmental and reproductive biology of different ferns and fern allies.

Differential proteomics of Picrorhiza kurrooa Royle ex Benth. in response to dark stress.

Picrorhiza (Picrorhiza kurrooa Royle ex Benth.) an important medicinal herb of western Himalayan region has been used to treat various diseases and disorders. Over-harvesting and lack of cultivation has led to its entry in Red Data Book as an endangered species. Further, its very restrictive habitat and lesser biomass production are major limitations for bringing it under commercial cultivation. All these issues necessitate deeper insights into mechanisms governing its growth and interaction with the environmental cues. Light may be one of the important factors to be studied for its role in regulating growth and adaptation of Picrorhiza as in natural habitat it prefers shady niches. Keeping this in view, proteome of Picrorhiza kept under light vis-à-vis under dark was analysed and compared. Leaf as well as root proteome of Picrorhiza was studied. Denaturing two dimensional gel electrophoresis and mass spectrometry techniques were used to detect and identify differentially expressed proteins, respectively. Twenty two proteins from leaf and 25 proteins from root showed differential expression levels under dark and light conditions. Among the differentially expressed proteins, majority were those involved in metabolism, protein synthesis, and stress and defense response. Other differentially expressed proteins were those involved in photosynthetic process, photorespiration and few proteins were with unknown function indicating that many different processes work together to establish a new cellular homeostasis in response to dark and light conditions. Proteins found to be differentially expressed under light vis-à-vis dark conditions suggested a range of biochemical pathways and processes being associated with response of plant to dark conditions. The identified proteins may be utilized for developing strategies for improving the biomass production/performance of Picrorhiza under varied light/dark habitats.

Osmotin-expressing transgenic tea plants have improved stress tolerance and are of higher quality.

Drought is a major stress that affects the yield and quality of tea, a widely consumed beverage crop grown in more than 20 countries of the world. Therefore, osmotin gene-expressing transgenic tea plants produced using earlier optimized conditions were evaluated for their tolerance of drought stress and their quality. Improved tolerance of polyethylene glycol-induced water stress and faster recovery from stress were evident in transgenic lines compared with the normal phenotype. Significant improvements in growth under in-vitro conditions were also observed. Besides enhanced reactive oxygen species-scavenging enzyme activity, the transgenic lines contained significantly higher levels of flavan-3-ols and caffeine, key compounds that govern quality and commercial yield of the beverage. The selected transgenic lines have the potential to meet the demands of the tea industry for stress-tolerant plants with higher yield and quality. These traits of the transgenic lines can be effectively maintained for generations because tea is commercially cultivated through vegetative propagation only.

Components of antioxidant system of Picrorhiza kurrooa exhibit different spatio-temporal behavior.

Antioxidant system is one of the important factors in regulating plant growth, development and adaptation. Thus, in order to have better insights into molecular mechanisms of growth and adaptation of a plant it is prerequisite to have known the status of various components of the antioxidant system of the plant. Here we studied the status of enzymatic and non-enzymatic components of the antioxidant system of picrorhiza (Picrorhiza kurrooa). Picrorhiza is an important medicinal herb of western Himalayan region and has been listed in the Red Data Book as an endangered species. Spatio-temporal analysis of ascorbic acid and glutathione in leaf, root and rhizome during different stages of development revealed differential status of these antioxidant molecules. Of the three tissues, ascorbic acid was found to be highest in leaves and lowest in roots. Interestingly, just opposite to that, glutathione was highest in roots and lowest in leaves. Using degenerate primers based approach followed by rapid amplification of complementary DNA (cDNA) ends method, full length cDNAs of three important genes namely Picrorhiza kurrooa ascorbate peroxidase (pkapx), Picrorhiza kurrooa monodehydroascorbate reductase (pkmdhar) and Picrorhiza kurrooa glutathione reductase (pkgr) of antioxidant system were cloned from picrorhiza. Complementary DNAs of pkapx, pkmdhar and pkgr contained 1,049, 2,016 and 1,664 bp, respectively. Expression analysis showed differential spatio-temporal expression of these genes. Expressions of all the three genes were found higher in roots as compared to rhizome and leaves. Temporal expression analysis of pkapx, pkmdhar and pkgr revealed differential transcript levels. Expression of pkapx exhibited negative correlation with the light intensity. Just opposite to the pkapx, expression pattern of pkgr revealed its positive correlation with light intensity. Expression pattern of pkmdhar revealed its light independent expression behavior. The findings may be useful to assess the role of cloned genes in picrorhiza growth, adaptation and can further be utilized for transgenic development for desired trait(s).

Genome-wide organization and expression profiling of the NAC transcription factor family in potato (Solanum tuberosum L.).

NAC [no apical meristem (NAM), Arabidopsis thaliana transcription activation factor [ATAF1/2] and cup-shaped cotyledon (CUC2)] proteins belong to one of the largest plant-specific transcription factor (TF) families and play important roles in plant development processes, response to biotic and abiotic cues and hormone signalling. Our genome-wide analysis identified 110 StNAC genes in potato encoding for 136 proteins, including 14 membrane-bound TFs. The physical map positions of StNAC genes on 12 potato chromosomes were non-random, and 40 genes were found to be distributed in 16 clusters. The StNAC proteins were phylogenetically clustered into 12 subgroups. Phylogenetic analysis of StNACs along with their Arabidopsis and rice counterparts divided these proteins into 18 subgroups. Our comparative analysis has also identified 36 putative TNAC proteins, which appear to be restricted to Solanaceae family. In silico expression analysis, using Illumina RNA-seq transcriptome data, revealed tissue-specific, biotic, abiotic stress and hormone-responsive expression profile of StNAC genes. Several StNAC genes, including StNAC072 and StNAC101that are orthologs of known stress-responsive Arabidopsis RESPONSIVE TO DEHYDRATION 26 (RD26) were identified as highly abiotic stress responsive. Quantitative real-time polymerase chain reaction analysis largely corroborated the expression profile of StNAC genes as revealed by the RNA-seq data. Taken together, this analysis indicates towards putative functions of several StNAC TFs, which will provide blue-print for their functional characterization and utilization in potato improvement.

Characterization of a T-DNA promoter trap line of Arabidopsis thaliana uncovers a cryptic bi-directional promoter.

Investigation of the transgenic Arabidopsis promoter trap line GFP-868 that showed GFP expression only in anthers revealed the T-DNA insertion at 461bp upstream to the hypothetical gene At4g10596 with the GFP reporter gene in head-to-head orientation to the At4g10596 gene. The expression of the At4g10596 gene in wild type and in GFP-868 plant homozygous for T-DNA insertion was comparable and found in all tissues tested, while the GFP expression was restricted to anthers of the GFP-868 plants suggesting that the 461bp fragment separating the two genes in the GFP-868 line is functioning as bi-directional promoter. This 461bp fragment was cloned upstream to the GUS gene in two orientations to test for bi-directional promoter activity. Transgenic Arabidopsis plants carrying either of these constructs showed GUS activity in anthers indicating that this fragment behaves as bi-directional promoter specific to anthers. These results were also supported by the presence of cis-acting motifs such as TATA box and POLLEN1LELAT52 (AGAAA) within the 461bp sequence in both orientations. However, transcripts corresponding to the upstream sequences beyond -461 nucleotides were not detected in the wild type suggesting that this 461bp fragment is a cryptic promoter. The significance of the promoter trap approach and the usefulness of this type of promoter are discussed.

Braving the attitude of altitude: Caragana jubata at work in cold desert of Himalaya.

The present work was conducted to understand the basis of adaptation in Caragana jubata in its niche environment at high altitude cold desert of Himalaya. Molecular data showed predominance of genes encoding chaperones and those involved in growth and development at low temperature (LT), a major cue operative at high altitude. Importantly, these genes expressed in C. jubata in its natural habitat. Their homologues in Arabidopsis thaliana, Oryza sativa, and Glycine max did not exhibit similar trend of gene expression at LT. Constitutive expression and a quick up-regulation of the above genes suggested the ability of C. jubata to adjust its cellular machinery to maintain growth and development in its niche. This was reflected in LT(50 )(the temperature at which 50% injury occurred) and LT mediated photosynthetic acclimatory response. Such molecular and physiological plasticity enables C. jubata to thrive in the high altitude cold desert of Himalayas.

Influence of human lactoferrin expression on iron homeostasis, flavonoids, and antioxidants in transgenic tobacco.

This study was aimed at to check the influence of human lactoferrin (hLF) expression on iron homeostasis, flavonoids, and antioxidants in transgenic tobacco. Transgenic tobacco expressing hLF cDNA under the control of a CaMV 35S promoter was produced. The iron content as well as chlorophyll content of transgenic tobacco was lower compared to mock and untransformed wild plants. Interestingly, hLF transgenic tobacco showed higher level of transcript expression for genes related to iron content regulation like iron transporter and metal transporter. While expression of genes related to iron storage such as ferritin 1 and ferritin 2 was downregulated. The transcript expression of genes encoding antioxidant enzymes such as glutathione reductase, glutathione-S-transferase, ascorbate peroxidase, and catalase was downregulated in hLF transgenic tobacco compared to controls. Further, the transcript expression of two important genes encoding dihydroflavonol reductase (DFR) and phenylalanine ammonia lyase regulatory enzymes of flavonoid biosynthesis pathway was analyzed. The expression of DFR was found to be downregulated, while PAL expression was upregulated in hLF transgenic tobacco compared to mock and untransformed wild plant. Total phenolics, flavonoids, and proanthocyanidins contents were found to be higher in hLF transgenic tobacco than the mock and untransformed wild plant. Results suggest that hLF expression in transgenic tobacco leads to iron deficiency, downregulation of antioxidant enzymes, and increase in total flavonoids.

Change in protein content during seed germination of a high altitude plant Podophyllum hexandrum Royle.

Podophyllum hexandrum Royle (=Sinopodophyllum hexandrum) is a high-altitude medicinal plant exploited for its etoposides which are potential anticancer compounds. An effective, conventional propagation method is by seed. However, seed germination is erratic, and seedling survival is low. A marginal increase in Podophyllum seed germination was attained with organic solvents. In the present study an attempt was made to decipher the physiological and biochemical barriers in terms of change in proteins during seed germination of Podophyllum. Comparative 2-DE analysis between un-germinated (dormant) and germinating seeds revealed nearly 113 differentially expressed proteins, whereas Peptide Mass Fingerprint (PMF) analysis of 97 protein spots revealed appearance of 27 proteins, up-accumulation of 11 proteins, down-accumulation of 19 proteins and disappearance of 40 proteins with germination. Identified 59 proteins in the homology search were involved in metabolism (carbohydrate and amino acid metabolism; 20 proteins), ABA/GA signaling (17 proteins) and stress (15 proteins) related proteins. Seven proteins were with unknown function. Two-DE, and MS/MS analysis in conjunction with semi-quantitative RT-PCR data of cell wall hydrolyzing genes, revealed that in Podophyllum the radicle protrusion occurs might be because of the up-accumulation of cell wall hydrolases i.e. ß-1, 3-glucanase and XET which weakens the thick walled micropylar endosperm.