Endosperm Tissue Research Articles

Zinc distribution and localization in primed maize seeds and its translocation during early seedling development

Zinc (Zn) priming is a technique used to increase seed Zn reserves for improving seed quality, crop growth, and enhancing stress tolerance in crop plants. The present study demonstrated the effect of water and Zn priming on the distribution and accumulation of endogenous and primed Zn in maize seeds (Zea mays L.). Zn concentration in unprimed, water and Zn primed seeds and germinated seedlings were analyzed by ICP-MS (Inductivity Coupled Plasma Mass Spectroscopy). DTZ (Diphenyle Thio-Carbazone) staining method and LA-ICP-MS (Laser Ablation Inductivity Coupled Plasma Mass Spectroscopy) scanning was used for showing Zn distribution and localization in the seeds. Zn priming significantly increased Zn concentration and content in seeds. Results of ICP-MS analysis showed a substantial increase in the testa and endosperm tissues after Zn priming. DTZ staining and LA-ICP-MS scanning of maize seeds revealed an uneven distribution of Zn in water and Zn primed seeds. Laser ablation data of water primed maize seeds demonstrated a significant (p<0.05) relocation of endogenous Zn from the aleurone layers towards the inner endosperm. Zn priming increased endosperm Zn content 3-fold compared with water primed seeds, while in the testa this increase was 50-fold. Furthermore, Zn priming significantly (p<0.05) increased the biomass of 10-d old maize seedlings grown in rolls of filter paper. Translocation of primed Zn towards shoots and roots was double than that of endogenous Zn in unprimed and water primed maize seeds. This is the first report of the distribution and accumulation of primed Zn in maize seeds. Further investigations are needed to understand the binding capacity of the different tissues within maize seeds and the retranslocation of primed Zn during early seedling development and plant growth.

Seed dormancy and germination

Reproduction is a critical time in plant life history. Therefore, genes affecting seed dormancy and germination are among those under strongest selection in natural plant populations. Germination terminates seed dispersal and thus influences the location and timing of plant growth. After seed shedding, germination can be prevented by a property known as seed dormancy. In practise, seeds are rarely either dormant or non-dormant, but seeds whose dormancy-inducing pathways are activated to higher levels will germinate in an ever-narrower range of environments. Thus, measurements of dormancy must always be accompanied by analysis of environmental contexts in which phenotypes or behaviours are described. At its simplest, dormancy can be imposed by the formation of a simple physical barrier around the seed through which gas exchange and the passage of water are prevented. Seeds featuring this so-called 'physical dormancy' often require either scarification or passage through an animal gut (replete with its associated digestive enzymes) to disrupt the barrier and permit germination. In other types of seeds with 'morphological dormancy' the embryo remains under-developed at maturity and a dormant phase exists as the embryo continues its growth post-shedding, eventually breaking through the surrounding tissues. By far, the majority of seeds exhibit 'physiological dormancy' - a quiescence program initiated by either the embryo or the surrounding endosperm tissues. Physiological dormancy uses germination-inhibiting hormones to prevent germination in the absence of the specific environmental triggers that promote germination. During and after germination, early seedling growth is supported by catabolism of stored reserves of protein, oil or starch accumulated during seed maturation. These reserves support cell expansion, chloroplast development and root growth until photoauxotrophic growth can be resumed.

Identification and quantification of flavonoids in yellow grain mutant of rice (Oryza sativa L.)

Flavonoids are naturally occurring phenolic compounds with potential health-promoting activities. Although anthocyanins and phenolic acids in coloured rice have been investigated, few studies have focused on flavonoids. Herein, we analysed flavonoids in a yellow grain rice mutant using UHPLC-DAD-ESI-Q-TOF-MS, and identified 19 flavonoids by comparing retention times and accurate mass measurements. Among them, six flavonoids, isoorientin, isoorientin 2″-O-glucoside, vitexin 2″-O-glucoside, isovitexin, isoscoparin 2″-O-glucoside and isoscoparin, were isolated and fully identified from the yellow grain rice mutant, and the levels were significantly higher than wild-type, with isoorientin particularly abundant in mutant embryo. Significant differences in total phenolic compounds and antioxidant activity were observed in mutant rice by DPPH, FRAP and TEAC assays. The results suggest that the representative six flavonoids may play an important role in colouration and antioxidant activity of embryo and endosperm tissue. The findings provide insight into flavonoid biosynthesis and the possibility of improving functionality in rice.

A Combination of Histological, Physiological, and Proteomic Approaches Shed Light on Seed Desiccation Tolerance of the Basal Angiosperm Amborella trichopoda.

Desiccation tolerance allows plant seeds to remain viable in a dry state for years and even centuries. To reveal potential evolutionary processes of this trait, we have conducted a shotgun proteomic analysis of isolated embryo and endosperm from mature seeds of Amborella trichopoda, an understory shrub endemic to New Caledonia that is considered to be the basal extant angiosperm. The present analysis led to the characterization of 415 and 69 proteins from the isolated embryo and endosperm tissues, respectively. The role of these proteins is discussed in terms of protein evolution and physiological properties of the rudimentary, underdeveloped, Amborella embryos, notably considering that the acquisition of desiccation tolerance corresponds to the final developmental stage of mature seeds possessing large embryos.

Seed development of Jatropha curcas L. (Euphorbiaceae): integrating anatomical, ultrastructural and molecular studies

This work provides a detailed histological analysis of the development of Jatropha curcas seeds, together with an assessment of the role of programmed cell death in this process. Seeds of Jatropha curcas are a potential source of raw material for the production of biodiesel, but very little is known about how the architecture of the seeds is shaped by the coordinated development of the embryo, endosperm and maternal tissues, namely integuments and nucellus. This study used standard anatomical and ultrastructural techniques to evaluate seed development and programmed cell death (PCD) in the inner integument was monitored by qPCR. In these studies, we found that the embryo sac formation is of the Polygonum type. We also found that embryogenesis is a slow process and the embryo is nourished by the suspensor at earlier stages and by nutrients remobilized from the lysis of the inner integument at later stages. Two types of programmed cell death contribute to the differentiation of the inner integument that begins at early stages of seed development. In addition, the mature embryo presents features of adaptation to dry environments such as the presence of four seminal roots, water absorbing stomata in the root zone and already differentiated protoxylem elements. The findings in this study fill in gaps related to the ontogeny of J. curcas seed development and provide novel insights regarding the types of PCD occurring in the inner integument.

Hormonal and morphological changes during seed development of Cypripedium japonicum.

With the goal of evaluating the factors that may be influencing the reproductive success of an endangered, terrestrial orchid Cypripedium japonicum Thunb., the anatomical and morphological characteristics of seed development were assessed, as were the levels of endogenous hormones to investigate the possible relationship between hormones and seed development. The results showed that morphological characteristics of seeds were changing from milky white and filiform to brown and fusiform. The development of ovule that initiated on the fertile valves only began after pollination, and a mature embryo sac was produced at 40days after pollination (DAP). Fertilization occurred 50 to 60DAP, and the newly formed zygote divided into two unequal cells that became the embryo proper and the suspensor. Seeds reached maturity at about 115DAP, at which point they consisted only of a globular embryo and two layers of seed coat, without endosperm. The suspensor was lost, and only lipid particles and a large number of starch grains distributed in the cytoplasm. The peak of gibberellic acid (GA3), zeatin riboside (ZR), and indole acetic acid (IAA) occurred in a time sequence, as 20, 30, and 40DAP. There was a highly significant positive correlation among them (P<0.01). Abscisic acid (ABA) content increased at 60DAP and reached the maximum at 115DAP. ABA contents were significantly negatively correlated with the three hormones above (P<0.05). It indicated that the lack of endosperm and suspensor tissues, delayed differentiation of embryos, and high levels of ABA may lead to the difficulty of germination.

Autoinoculation trap for management of Hypothenemus hampei (Ferrari) with Beauveria bassiana (Bals.) in coffee crops

An autoinoculation trap containing the entomopathogenic fungus Beauveria bassiana (Bals.) was developed for the management of the coffee berry borer, Hypothenemus hampei (Ferrari). The trap is composed of a plastic cylindrical jar with inlet holes in the upper part and outlet holes at the bottom. Within the upper part of the trap, we placed a diffusion vial filled with a 1:1 mixture of ethanol and methanol as an attractant for adult H. hampei. In the bottom part, we lined the surfaces of the two exit holes with a strip of woven fabric (wool) impregnated with an average of 5.44±0.33×108conidia.cm−2 of the B. bassiana isolate ESALQ-PL63. A five-second exposure of female coffee berry borers to the fungus-impregnated fabric resulted in the acquisition of 2.9±0.77×105conidia per insect and 88.5% confirmed mortality rate in the laboratory. Although most of beetles that were contaminated in the autoinoculation trap in the laboratory were able to penetrate the coffee berry exocarp (the outermost layer), only 5% reached the endosperm (tissue that surrounds and provides nutrition to the embryo). This indicates that very little damage to coffee seeds is expected from borers that pass through the autoinoculation traps. Additionally, these traps provided high levels of H. hampei mortality in the field. The confirmed mortalities at the end of the first and second field trials after 151 and 40days were 66.3±5.2% and 92.2±4.7%, respectively. Altogether, these results indicate that autoinoculation traps containing B. bassiana have great potential to be used in the integrated management of H. hampei.

Spatiotemporal modulation of abscisic acid and gibberellin metabolism and signalling mediates the effects of suboptimal and supraoptimal temperatures on seed germination in wheat (Triticum aestivum L.).

Seed germination is a complex process regulated by intrinsic hormonal cues such as abscisic acid (ABA) and gibberellin (GA), and environmental signals including temperature. Using pharmacological, molecular and metabolomics approaches, we show that supraoptimal temperature delays wheat seed germination through maintaining elevated embryonic ABA level via increased expression of ABA biosynthetic genes (TaNCED1 and TaNCED2), increasing embryo ABA sensitivity through upregulation of genes regulating ABA signalling positively (TaPYL5, TaSnRK2, ABI3 and ABI5) and decreasing embryo GA sensitivity via induction of TaRHT1 that regulates GA signalling negatively. Endospermic ABA and GA appeared to have minimal roles in regulating germination at supraoptimal temperature. Germination inhibition by suboptimal temperature is associated with elevated ABA level in the embryo and endosperm tissues, mediated by induction of TaNCEDs and decreased expression of endospermic ABA catabolic genes (TaCYP707As), and increased ABA sensitivity in both tissues via upregulation of TaPYL5, TaSnRK2, ABI3 and ABI5 in the embryo and TaSnRK2 and ABI5 in the endosperm. Furthermore, suboptimal temperature suppresses GA synthesis in both tissues and GA sensitivity in the embryo via repressing GA biosynthetic genes (TaGA20ox and TaGA3ox2) and inducing TaRHT1, respectively. These results highlight that spatiotemporal modulation of ABA and GA metabolism and signalling in wheat seeds underlies germination response to temperature.

Using ICP and micro-PIXE to investigate possible differences in the mineral composition of genetically modified versus wild-type sorghum grain

In the present study, possible differences in the mineral composition of transgenic versus non-transgenic sorghum grains were investigated using inductively coupled atomic emission spectroscopy (ICP-AES); and, in-tissue elemental mapping by micro Proton-Induced X-ray Emission (micro-PIXE) analysis. ICP AES was used to analyse the bulk mineral content of the wholegrain flour derived from each genotype; whilst micro-PIXE was used to interrogate localised differences in mineral composition specific to certain areas of the grain, such as the bran layer and the central endosperm tissue. According to the results obtained, no significant difference in the average Fe, Zn or Ca content was found to differentiate the transgenic from the wild-type grain using ICP-AES. However, using micro-PIXE, a significant reduction in zinc could be detected in the bran layer of the transgenic grains relative to wild-type. Although it is difficult to draw firm conclusions, as a result of the small sample size used in this study, micro-PIXE has nonetheless proven itself as a useful technique for highlighting the possibility that there may be reduced levels of zinc accumulation in the bran layer of the transgenic grains. Given that the genetic modification targets proteins that are highly concentrated in certain parts of the bran tissue, it seems plausible that the reduced levels of zinc may be an unintended consequence of the silencing of kafirin proteins. Although no immediate health or nutritional concerns emerge from this preliminary finding, it is noted that zinc plays an important biological role within this part of the grain as a structural stabiliser and antioxidant factor. Further study is therefore needed to assess more definitively the extent of the apparent localised reduction in zinc in the transgenic grains and how this may affect other important grain quality characteristics.

E+ subgroup PPR protein defective kernel 36 is required for multiple mitochondrial transcripts editing and seed development in maize and Arabidopsis.

Mitochondria are semi-autonomous organelles that are the powerhouse of the cells. Plant mitochondrial RNA editing guided by pentatricopeptide repeat (PPR) proteins is essential for energy production. We identify a maize defective kernel mutant dek36, which produces small and collapsed kernels, leading to embryos and/or seedlings lethality. Seed filling in dek36 is drastically impaired, in line with the defects observed in the organization of endosperm transfer tissue. Positional cloning reveals that DEK36, encoding a mitochondria-targeted E+ subgroup PPR protein, is required for mitochondrial RNA editing at atp4-59, nad7-383 and ccmFN -302, thus resulting in decreased activities of mitochondrial complex I, complex III and complex IV in dek36. Loss-of-function of its Arabidopsis ortholog At DEK36 causes arrested embryo and endosperm development, leading to embryo lethality. At_dek36 also has RNA editing defects in atp4, nad7, ccmFN1 and ccmFN2 , but at the nonconserved sites. Importantly, efficiency of all editing sites in ccmFN1 , ccmFN2 and rps12 is severely decreased in At_dek36, probably caused by the impairment of their RNA stabilization. These results suggest that the DEK36 orthologue pair are essential for embryo and endosperm development in both maize and Arabidopsis, but through divergent function in regulating RNA metabolism of their mitochondrial targets.

Applying the INTACT method to purify endosperm nuclei and to generate parental-specific epigenome profiles.

The early endosperm tissue of dicot species is very difficult to isolate by manual dissection. This protocol details how to apply the INTACT (isolation of nuclei tagged in specific cell types) system for isolating early endosperm nuclei of Arabidopsis at high purity and how to generate parental-specific epigenome profiles. As a Protocol Extension, this article describes an adaptation of an existing Nature Protocol that details the use of the INTACT method for purification of root nuclei. We address how to obtain the INTACT lines, generate the starting material and purify the nuclei. We describe a method that allows purity assessment, which has not been previously addressed. The purified nuclei can be used for ChIP and DNA bisulfite treatment followed by next-generation sequencing (seq) to study histone modifications and DNA methylation profiles, respectively. By using two different Arabidopsis accessions as parents that differ by a large number of single-nucleotide polymorphisms (SNPs), we were able to distinguish the parental origin of epigenetic modifications. Our protocol describes the only working method to our knowledge for generating parental-specific epigenome profiles of the early Arabidopsis endosperm. The complete protocol, from silique collection to finished libraries, can be completed in 2 d for bisulfite-seq (BS-seq) and 3 to 4 d for ChIP-seq experiments.This protocol is an extension to: Nat. Protoc. 6, 56-68 (2011); doi:10.1038/nprot.2010.175; published online 16 December 2010.

Research Article Differential expression of dormancy-Associated genes in fastigiata and hypogaea peanut

Seed dormancy is a temporary replacement state observed in some higher plants, which prevents germination under unfavorable conditions. In several species, dormancy process is triggered by hormonal factors, initiated by ABA and precursor enzymes. In this work, we investigated the relationship between dormancy and the expression of ARP, DMR1, and NCED genes in upright and runner peanut seeds. Eight dormancy-contrasting genotypes were previously phenotyped based on germination traits, during three storage periods. Expression of ARP, DMR1 and NCED transcripts was analyzed by qRT-PCR, from endosperm and embryo tissues. Higher expression of NCED and ARP were found in embryo tissues from runner genotypes, IAC Caiapo and LGoPE-06, confirming findings in literature as to association of these genes with seed dormancy. Higher expression of DMR1 was found only in endosperm of upright materials (L7 Bege and BR1). We suggest the NCED is a functional molecular marker to identify dormancy in seeds, contributing to selection procedures of runner genotypes in peanut breeding.

Genetic Imprinting: Comparative Analysis Between Plants and Mammals

Genetic imprinting: the parent of origin?specific biased expression of alleles is an important type of epigenetic gene regulation in flowering plants and mammals. All imprinted genes show either maternal ? or paternal?specific mono?allelic expression. Considering that plants and mammals shared a common ancestor more than one billion years ago, significant overlap and potentially equally significant differences in the genomic imprinting mechanisms in these two taxa are emerging. In plants, the imprinted genes are primarily imprinted in the ephemeral endosperm tissues of the seeds which do not contribute any genome to future generations, while in mammals, the imprinted genes are located in embryo, placenta, and the adult body. Though both kingdoms silence imprinted genes using DNA methylation, imprinted alleles in mammals are targeted for silencing while in plants preexisting methylation is specifically removed from the allele destined to be active in maternally expressed genes in the endosperm. It is now accepted that imprinting evolved in both taxa due to competition between parental genomes over resource allocation to offspring. Moreover, the distinct life cycle stages between the taxa may account for the different strategies used by plants and mammals to regulate parent?specific gene expression. The elucidation of the genetic basis and molecular mechanisms responsible for genetic imprinting have provided answers to various crucial questions arising in biological sciencesPlant Tissue Cult. &amp; Biotech. 26(2): 267-284, 2016 (December)

Accumulation characteristic of protein bodies in different regions of wheat endosperm under drought stress

The structural characteristics of protein body accumulation in different endosperm regions of hard wheat cultivar (XM33) and soft wheat cultivar (NM13) under drought stress were investigated. Drought stress treatment was implemented from plant regreening to the caryopsis mature stage. Microscope images of endosperm cells were obtained using resin semithin slice technology to observe the distribution and relative area of protein body (PB). Compared with NM13, relative PB area of XM33 was significantly higher in sub-aleurone endosperm region. The amount of accumulation, including the size and relative area of PB, in two wheat cultivars was higher in sub-aleurone region than that in central region at 18 days post anthesis (DPA). Drought stress significantly enhanced the sizes and relative areas of PBs in the dorsal and abdominal endosperms in two wheat cultivars. Particularly for dorsal endosperm, drought stress enhanced the relative PB area at 18 DPA and NM13 (5.0% vs. 6.73%) showed less enhancement than XM33 (5.49% vs. 8.96%). However, NM13 (9.58% vs. 12.02%) showed greater enhancement than XM33 (10.25% vs. 11.7%) at 28 DPA. The protein content in the dorsal and abdominal endosperms of the two wheat cultivars decreased at 12 DPA and then increased until 38 DPA. Drought stress significantly increased the protein contents in the two main regions. From 12 to 38 DPA, the amount of PB accumulation and the protein content were higher in XM33 than those in NM13. The results revealed that PB distribution varied in different endosperm tissues and that the amount of PB accumulation was remarkably augmented by drought stress.

A mutational approach for the detection of genetic factors affecting seed size in maize.

Genes influencing seed size. The designation emp (empty pericarp) refers to a group of defective kernel mutants that exhibit a drastic reduction in endosperm tissue production. They allow the isolation of genes controlling seed development and affecting seed size. Nine independently isolated emp mutants have been analyzed in this study and in all cases longitudinal sections of mature seeds revealed the absence of morphogenesis in the embryo proper, an observation that correlates with their failure to germinate. Complementation tests with the nine emp mutants, crossed inter se in all pairwise combinations, identified complementing and non-complementing pairs in the F1 progenies. Data were then validated in the F2/F3 generations. Mutant chromosomal location was also established. Overall our study has identified two novel emp genes and a novel allele at the previously identified emp4 gene. The introgression of single emp mutants in a different genetic background revealed the existence of a cryptic genetic variation (CGV) recognizable as a variable increase in the endosperm tissue. The unmasking of CGV by introducing single mutants in different genetic backgrounds is the result of the interaction of the emp mutants with a suppressor that has no obvious phenotype of its own and is present in the genetic background of the inbred lines into which the emp mutants were transferred. On the basis of these results, emp mutants could be used as tools for the detection of genetic factors that enhance the amount of endosperm tissue in the maize kernel and which could thus become valuable targets to exploit in future breeding programs.

Small family, big impact: In silico analysis of DREB2 transcription factor family in rice

Dehydration-responsive element- (DREB) proteins are considered as the master regulators of plant abiotic stress responses including drought, salinity and cold. They are also involved in other developmental processes such as embryo and endosperm development. DREB family of transcription factors consist of two sub families namely CBF1/DREB1 and DREB2. In this study, a genome-wide in silico analysis was carried out to dissect the structure and function of DREB2 family transcription factors in the rice genome. Using Arabidopsis DREB2 sequences a total of five rice DREB2 homologs were identified and they were distributed among four chromosomes. All OsDREBs contained the AP2 domain and unique [K/R]GKKGPxN motif characteristic to DREB2 family. During rice growth and development, three OsDREB2s namely OsDREB2A, OsDREB2B and OsABI4 were expressed and their expression was confined to embryo and endosperm tissues. OsDREB2A, OsDREB2B and OsDREB2C were expressed under abiotic stress conditions. OsDREB2B was expressed under drought, salinity and cold stress conditions while OsDREB2A and OsDREB2C were expressed only under drought and salinity conditions. The putative promoter regions of OsDREB2s were enriched with elements related to cellular development, hormonal regulation and stress response validating the observed expression dynamics. Co-expression analysis revealed that embryo development and stress related genes were expressed together with OsDREB2s. Predicted post-translational modifications indicated the fine regulation of OsDREB2s. These findings may shed light in uncovering the complex abiotic stress signaling networks and future genomics studies targeting the development of climate ready crops.

Variation of starch granules in diploid species of the genus Avena L.

Composite starch granules, the main product of assimilation in oat endosperm, were analyzed in the accessions of both wild and cultivated diploid species of the genus Avena. Simple starch granules are mostly synthesized in the outer parts of the endosperm tissue. The size of sub-grains in a composite granule does not depend on a wild or cultivated status of the species. Inter-specific variation in the size differences of composite granules is large. Also, a broad variation has been detected for granules analyzed in a Lugol’s solution or polarizing light. This analysis revealed a difference between the synthesis of amylopectin versus amylose in a granule. Examples of occurrences of low levels of amylopectin synthesis are provided.

Cover Caption: Rice Caryopsis Development

As one of the most important food sources, rice caryopsis has a complex structure that composes of maternal tissues of pericarp and testa, and filial tissues of endosperm and embryo. In this issue, two papers, authored by Wu et al, illustrate in detail the development of maternal (pp. 772–785) and endosperm tissues (pp. 786–798). Results provided show that rice caryopsis undergoes dynamic changes in mitotic divisions, cellularization, cell differentiation and programmed cell death.

Cloning and characterization of TaMBD6 homeologues encoding methyl-CpG-binding domain proteins in wheat

DNA methylation is a major epigenetic marker in plants that plays a crucial role in transcriptional and developmental regulation. The DNA methylation ‘code’ is thought to be ‘read’ by a set of proteins containing methyl-CpG-binding domain (MBD). However, little is known about MBD genes in common wheat (Triticum aestivum L.). Here, we report the isolation and characterization of TaMBD6 and its homeologues (TaMBD6_A, TaMBD6_B, and TaMBD6_D) in hexaploid wheat. The cDNA was quite different among the three homeologues and InDel mutations were detected in 5′-UTR and coding region. Two types of TRs (tandem repeats) -- TR1 (57 bp) and TR2 (39 bp) -- occurred in the coding region. TaMBD6_B harbored five copies of TR1 and two copies of TR2. In contrast, TaMBD6_A lacked 30 bp between the 2nd and 3rd copy of TR1, while TaMBD6_D was missing two copies of TR1 but had three copies of TR2. TaMBD6_A, TaMBD6_B, and TaMBD6_D encoded 435, 446, and 420 amino acids, respectively. Structural analysis of TaMBD6 protein indicated that each of the three homeologues had an identical MBD domain at the N-terminal, as well as a typical nuclear localization signal. Although genomics analysis showed that two introns were included, the length of the first intron varied from 3100 bp to 3471 bp and their sequences were very different. Expression analysis demonstrated that the transcription level of TaMBD6 began to increase gradually in developing grains at 15 days after pollination while decreasing significantly in endosperm and embryo tissues during germination. Expression of TaMBD6 appeared to be positively correlated with starch metabolism in the endosperm but was negatively correlated with embryo formation and sprouting. We were also interested to learn that TaMBD6 homeologues were differentially expressed in developing wheat plants and that their expression patterns were variously affected by vernalization treatment. Further investigation revealed that TaMBD6 was induced by prolonged chilling, indicating that the protein is potentially involved in regulating the developmental transition from vegetative to reproductive stages. Although the homeologues generally showed similar differential expression patterns, TaMBD6_D and TaMBD6_B contribute more to the processes of grain development and germination while TaMBD6_A is predominant in mature plants.

Characterization of Endosperm Carbohydrates in isa2–339 Maize and Interactions with su1‐ref

Selection for genetic modifiers of starch and sugar accumulation in sugary1‐ref (su1‐ref) maize (Zea mays L.) has led to the improvement of flavor characteristics in su1 sweet corn hybrids. The Su1 gene encodes a starch debranching enzyme that when defective causes large quantities of water‐soluble polysaccharides (WSP) to be produced in maize endosperm tissue. Another gene in maize, Isoamylase2 (Isa2), produces a starch debranching enzyme known to interact with the wild‐type SU1 protein in developing maize endosperm. Experiments were conducted to determine if a mutant of the Isa2 gene significantly modifies carbohydrate composition in su1‐ref inbreds and therefore is potentially useful for modifying flavor characteristics of su1 sweet corn. Using isa2–339, a mutant at the Isa2 locus, a population was created in which the su1‐ref and isa2–339 alleles were segregating. Lines developed from this population were used for single kernel carbohydrate analysis in 2012 and carbohydrate analysis of replicated ears in 2013. In 2012 and 2013, individual kernels and replicated ears fixed for the su1‐ref and isa2–339 alleles contained less starch and more total sugar compared with the su1‐ref, Isa2 parent, which was the source of the su1‐ref allele in this population. When the su1‐ref, isa2–339 mutant lines were reciprocally crossed with the Su1, isa2–339 parent, starch and WSP contents were significantly different among the Su1, isa2–339 parent and the hybrids. Kernels of both types of reciprocal hybrids had more WSP and less starch than did the Su1, isa2–339 parent. Kernels with two doses of the su1‐ref allele have more WSP and less starch than kernels with a single dose of the su1‐ref allele. In this population, starch content is further reduced in su1‐ref, isa2–339 double mutant genotypes compared with the su1‐ref parent. The su1‐ref/su1‐ref, isa2–339/isa2–339 lines developed in this experiment are the first documented double mutants of su1‐ref with another isoamylase starch debranching enzyme mutant.