Coordinated regulation of starch synthesis in maize endosperm by microRNAs and DNA methylation.

Starch synthesis is an essential feature of crop filling, but knowledge of the molecular mechanisms regulating the expression of starch synthesis genes (SSGs) is currently limited to transcription factors (TFs). Here, we obtained transcriptome, small RNAome, and DNA methylome data from maize (Zea mays) endosperms during multiple developmental stages and established a regulatory network atlas of starch synthesis. Transcriptome analysis showed a sharp transition at 9-10 days after pollination, when genes involved in starch and sucrose metabolism are upregulated and starch accumulates rapidly. Expression pattern analysis established a comprehensive network between SSGs and TFs. During maize endosperm development, the miRNAs with preferential repression of the expression of TFs, particularly the TFs regulating SSG expression, were extensively downregulated. Specifically, ZmMYB138 and ZmMYB115 affected the transcriptional activities of Du1/Wx and Ae1/Bt2 genes at their respective promoter regions. Remarkably, the two TFs were negatively regulated by the copious expression of Zma-miR159k-3p at the post-transcriptional level. This suggests that miRNAs are important regulators of starch synthesis. Moreover, with the exclusion of the TFs, the expression of both SSGs and miRNAs was globally regulated by DNA methylation. Altogether, the present results (i) establish the regulatory functions of miRNAs and DNA methylation in starch synthesis and (ii) indicate that DNA methylation functions as a master switch.

Genome-Wide Identification of DOF Gene Family and the Mechanism Dissection of SbDof21 Regulating Starch Biosynthesis in Sorghum.

Starch is one of the main utilization products of sorghum (Sorghum bicolor L.), the fifth largest cereal crop in the world. Up to now, the regulation mechanism of starch biosynthesis is rarely documented in sorghum. In the present study, we identified 30 genes encoding the C2-C2 zinc finger domain (DOF), with one to three exons in the sorghum genome. The DOF proteins of sorghum were divided into two types according to the results of sequence alignment and evolutionary analysis. Based on gene expressions and co-expression analysis, we identified a regulatory factor, SbDof21, that was located on chromosome 5. SbDof21 contained two exons, encoding a 36.122 kD protein composed of 340 amino acids. SbDof21 co-expressed with 15 genes involved in the sorghum starch biosynthesis pathway, and the Pearson correlation coefficients (PCCs) with 11 genes were greater than 0.9. The results of qRT-PCR assays indicated that SbDof21 is highly expressed in sorghum grains, exhibiting low relative expression levels in the tissues of roots, stems and leaves. SbDOF21 presented as a typical DOF transcription factor (TF) that was localized to the nucleus and possessed transcriptional activation activity. Amino acids at positions 182-231 of SbDOF21 formed an important structure in its activation domain. The results of EMSA showed that SbDOF21 could bind to four tandem repeats of P-Box (TGTAAAG) motifs in vitro, such as its homologous proteins of ZmDOF36, OsPBF and TaPBF. Meanwhile, we also discovered that SbDOF21 could bind and transactivate SbGBSSI, a key gene in sorghum amylose biosynthesis. Collectively, the results of the present study suggest that SbDOF21 acts as an important regulator in sorghum starch biosynthesis, exhibiting potential values for the improvement of starch contents in sorghum.

Genome-wide characterization of non-reference transposons in crops suggests non-random insertion.

BACKGROUND: Transposons (transposable elements or TEs) are DNA sequences that can change their position within the genome. A large number of TEs have been identified in reference genome of each crop(named accumulated TEs), which are the important part of genome. However, whether there existed TEs with different insert positions in resequenced crop accession genomes from those of reference genome (named non-reference transposable elements, non-ref TEs), and what the characteristics (such as the number, type and distribution) are. To identify and characterize crop non-ref TEs, we analyzed non-ref TEs in more than 125 accessions from rice (Oryza sativa), maize (Zea mays) and sorghum (Sorghum bicolor) using resequenced data with paired-end mapping methods. RESULTS: We identified 13,066, 23,866 and 35,679 non-ref TEs in rice, maize and sorghum, respectively. Genome-wide characterization analysis shows that most of non-ref TEs were unique and non-ref TE classes shows different among rice, maize and sorghum. We found that non-ref TEs have a strong positive correlation with gene number and have a bias toward insertion near genes, but with a preference for avoiding coding regions in maize and sorghum. The genes affected by non-ref TE insertion were functionally enriched for stress response mechanisms in all three crops. CONCLUSIONS: These observations suggest that transposon insertion is not a random event and it makes genomic diversity, which may affect the intraspecific adaption and evolution of crops.

Pleiotropic ZmICE1 Is an Important Transcriptional Regulator of Maize Endosperm Starch Biosynthesis.

Starch, the major component of cereal grains, affects crop yield and quality and is widely used in food and industrial applications. The biosynthesis of maize starch is a complex process involving a series of functional enzymes. However, the sophisticated regulatory mechanisms of starch biosynthetic genes have not been fully elaborated. The basic/helix-loop-helix (bHLH) transcription factors are widely distributed in eukaryotes and participate in many physiological processes. In this study, 202 bHLH encoding genes were identified in the maize genome by Blast method. ZmICE1 gene, which belongs to the ICE subfamily of the bHLH family, was obtained and expressed mainly in maize filling endosperm and co-expressed with 14 starch biosynthesis genes. Based on the comparative analyses across different plant species, we revealed that the gene structures and protein domains of the ICE subfamily were conserved between monocots and dicots, suggesting their functional conservation feature. Yeast activation and subcellular localization assays suggested that ZmICE1 had transcriptional activation activity and localized in the nucleus. Yeast one-hybrid assays confirmed that ZmICE1 could directly bind to the promoters of ZmSSIIa and ZmGBSSI. Transient gene expression analysis in maize endosperm revealed that ZmICE1 positively regulated the expression of ZmSSIIa, but inhibited the expression of ZmGBSSI. Our results indicated that ZmICE1 could function as a regulator of maize starch biosynthesis.

Profiling of transcriptional regulators associated with starch biosynthesis in sorghum (Sorghum bicolor L.).

Starch presents as the major component of grain endosperm of sorghum (Sorghum bicolor L.) and other cereals, serving as the main energy supplier for both plants and animals, as well as important industrial raw materials of human beings, and was intensively concerned world widely. However, few documents focused on the pathway and transcriptional regulations of starch biosynthesis in sorghum. Here we presented the RNA-sequencing profiles of 20 sorghum tissues at different developmental stages to dissect key genes associated with sorghum starch biosynthesis and potential transcriptional regulations. A total of 1,708 highly expressed genes were detected, namely, 416 in grains, 736 in inflorescence, 73 in the stalk, 215 in the root, and 268 genes in the leaf. Besides, 27 genes encoded key enzymes associated with starch biosynthesis in sorghum were identified, namely, six for ADP-glucose pyrophosphorylase (AGPase), 10 for starch synthases (SSs), four for both starch-branching enzymes (SBE) and starch-debranching enzymes (DBEs), two for starch phosphorylases (SPs), and one for Brittle-1 (BT1). In addition, 65 transcription factors (TFs) that are highly expressed in endosperm were detected to co-express with 16 out of 27 genes, and 90 cis-elements were possessed by all 27 identified genes. Four NAC TFs were cloned, and the further assay results showed that three of them could in vitro bind to the CACGCAA motif within the promoters of SbBt1 and SbGBSSI, two key genes associated with starch biosynthesis in sorghum, functioning in similar ways that reported in other cereals. These results confirmed that sorghum starch biosynthesis might share the same or similar transcriptional regulations documented in other cereals, and provided informative references for further regulatory mechanism dissection of TFs involved in starch biosynthesis in sorghum.

A design optimized prime editor with expanded scope and capability in plants.

The ability to manipulate the genome in a programmable manner has illuminated biology and shown promise in plant breeding. Prime editing, a versatile gene-editing approach that directly writes new genetic information into a specified DNA site without requiring double-strand DNA breaks, suffers from low efficiency in plants1-5. In this study, N-terminal reverse transcriptase-Cas9 nickase fusion performed better in rice than the commonly applied C-terminal fusion. In addition, introduction of multiple-nucleotide substitutions in the reverse transcriptase template stimulated prime editing with enhanced efficiency. By using these two methods synergistically, prime editing with an average editing frequency as high as 24.3% at 13 endogenous targets in rice transgenic plants, 6.2% at four targets in maize protoplasts and 12.5% in human cells was achieved, which is two- to threefold higher than the original editor, Prime Editor 3. Therefore, our optimized approach has potential to make more formerly non-editable target sites editable, and expands the scope and capabilities of prime editing in the future.

Environment-Driven Adaptations of Leaf Cuticular Waxes Are Inheritable for Medicago ruthenica.

Cuticular waxes covering the plant surface play pivotal roles in helping plants adapt to changing environments. However, it is still not clear whether the responses of plant cuticular waxes to their growing environments are inheritable. We collected seeds of Medicago ruthenica (a perennial legume) populations from 30 growing sites in northern China and examined the variations of leaf cuticular waxes in a common garden experiment. Four wax genes, MrFAR3-1, MrFAR3-2, MrCER1, and MrKCS1, involved in biosynthesis of predominant wax classes (primary alcohol and alkane) and wax precursors, were isolated to test the contributions of genetic variations of the coding sequences (CDS) and the promoter sequences and epigenetic modifications. The plasticity responses of the cuticular waxes were further validated by two stress-modeling experiments (drought and enhancing ultraviolet B). Great variations in total wax coverage and abundance of wax classes or wax compounds were observed among M. ruthenica populations in a common garden experiment. Stress-modeling experiments further validated that M. ruthenica would alter leaf wax depositions under changed growing conditions. The transcriptional levels of the wax genes were positively or negatively correlated with amounts of cuticular waxes. However, the analysis of promoter methylation showed that the methylation level of the promoter region was not associated with their expressions. Although both promoter sequences and CDS showed a number of polymorphic sites, the promoters were not naturally selected and insignificant difference could be observed in the numbers and types of acting elements of the four wax genes among populations. In contrast, the CDS of the wax genes were naturally selected, with a number of missense mutations resulting in alterations of the amino acid as well as their isoelectric points and polarities, which could impact on enzyme function/activity. We conclude that long-term adaptation under certain environments would induce genetic mutation of wax biosynthesis genes, resulting in inheritable alterations of cuticular wax depositions.

Responses of cuticular waxes of faba bean to light wavelengths and selection of candidate genes for cuticular wax biosynthesis.

Cuticular waxes play important eco-physiological roles in protecting plants against abiotic and biotic stresses and show high sensitivity to environmental changes. In order to clarify the responses of cuticular waxes on faba bean (Vicia faba L.) leaves to different light wavelengths, the phenotypic plasticity of cuticular waxes was analyzed when plants were subjected to white, red, yellow, blue, and purple light. Leaf samples from yellow, purple, and white lights were further analyzed, and candidate genes of wax biosynthesis were selected by RNA-seq technology and transcriptome processing. Yellow light increased the total wax coverage and changed the crystal structure compared with leaves under white light. Light wavelengths changed the relative abundance of dominant primary alcohol from C24 under white, yellow, and red lights to C26 under blue and purple lights. In total, 100,194 unigenes were obtained, and 10 genes were annotated in wax biosynthesis pathway, including VLCFAs elongation (KCS1, KCS4, LACS2 and LACS9), acyl reduction pathway (FAR3 and WSD1), and decarboxylation pathway (CER1, CER3 and MAH1). qRT-PCR analysis revealed that yellow and purple lights significantly influenced the expression levels of these genes. Yellow light also increased the water loss rate and decreased the photosynthesis rate. Light at different wavelengths particularly yellow light induced the changes of phenotypic plasticity of cuticular waxes, which thus altered the leaf eco-physiological functions. The expression levels of genes related to wax biosynthesis were also altered by different light wavelengths, suggesting that light at different wavelengths may also be applied in selecting candidate genes involved in wax biosynthesis in other crops.

Comparative analyses of cuticular waxes on various organs of faba bean (Vicia faba L.).

Cuticular waxes cover the plant surface and serve as hydrophobic layer, exhibiting various wax profiles between plant species and plant organs. This paper reports comprehensive analysis of the waxes on organs exposed to air, including leaf, stem, pod pericarp, and petals (banner, wing and keel), and on seed coat enwrapped in pod pericarp of faba bean (Vicia faba). In total 7 classes of wax compounds were identified, including fatty acids, primary alcohols, alkyl esters, aldehydes, alkanes, cinnamyl alcohol esters, and alkylresorcinols. Overall, primary alcohols dominated the waxes on leaves and the seed coat enwrapped in pod pericarp, alkanes accumulated largely in stem and petals, whereas alkylresorcinols were observed in leaf, stem and pod pericarp. Organs exposed to air had higher coverage (>1.2 µg/cm2) than those on seed coat (<0.8 µg/cm2), and keel having the highest wax coverage. Meanwhile, the wax coverage on seed coat reduced during the seed development. The variations of wax coverages, compound class distributions and chain length profiles among organs suggested that wax depositions were associated with their ecophysiological functions, and the enzymes involved in wax biosynthesis also showed organ-specific.

Responses of Phaseolus calcaltus to lime and biochar application in an acid soil.

INTRODUCTION: Rice bean (Phaseolus calcaltus), as an annual summer legume, is always subjected to acid soils in tropical to subtropical regions, limiting its growth and nodulation. However, little is known about its responses to lime and biochar addition, the two in improving soil fertility in acid soils. MATERIALS AND METHODS: In the current study, a pot experiment was conducted using rice bean on a sandy yellow soil (Orthic Acrisol) with a pH of 5.5. The experiment included three lime rates (0, 0.75 and 1.5 g kg-1) and three biochar rates (0, 5 and 10 g kg-1). The biochar was produced from aboveground parts of Solanum tuberosum using a home-made device with temperature of pyrolysis about 500 °C. RESULTS AND DISCUSSION: The results indicated that both lime and biochar could reduce soil exchange Al concentration, increase soil pH and the contents of soil microbial biomass carbon and microbial biomass nitrogen, and enhance urease and dehydrogenase activities, benefiting P. calcaltus growth and nodulation in acid soils. Lime application did decrease the concentrations of soil available phosphorus (AP) and alkali dispelled nitrogen (AN), whereas biochar application increased the concentrations of soil AP, AN and available potassium (AK). However, sole biochar application could not achieve as much yield increase as lime application did. High lime rate (1.5 g lime kg-1) incorporated with low biochar rate (5 g biochar kg-1) could obtain higher shoot biomass, nutrient uptake, and nodule number when compared with high lime rate and high biochar rate. CONCLUSION: Lime incorporated with biochar application could achieve optimum improvement for P. calcaltus growing in acid soils when compared with sole lime or biochar addition.

Identification and functional analysis of the ICK gene family in maize.

Inhibitors of cyclin-dependent kinases (ICKs) are key regulators of cyclin-dependent kinase activities and cell division. Herein, we identified eight ICKs in maize, which we named Zeama;ICKs (ZmICKs). Primary sequencing and phylogenetic analyses were used to divide the ZmICK family into two classes: group B and group C. Subcellular localization analysis of ZmICK:enhanced green fluorescent protein (eGFP) fusion constructs in tobacco leaf cells indicated that ZmICKs are principally nuclear. Co-localization analysis of the ZmICKs and maize A-type cyclin-dependent kinase (ZmCDKA) was also performed using enhanced green fluorescent protein (eGFP) and red fluorescent protein (RFP) fusion constructs. The ZmICKs and ZmCDKA co-localized in the nucleus. Semi-quantitative RT-PCR analysis of the ZmICKs showed that they were expressed at different levels in all tissues examined and shared similar expression patterns with cell cycle-related genes. Yeast two-hybrid and bimolecular fluorescence complementation assays showed that ZmICK1, ZmICK2, ZmICK3, and ZmICK4 interact with ZmCDKA1 and ZmCDKA3. Interestingly, ZmICK7 interacts with D-type cyclins. Transformed and expressed ZmCDKA1 and ZmICKs together in fission yeast revealed that ZmICK1, ZmICK3, and ZmICK4 can affect ZmCDKA1 function. Moreover, the C-group of ZmICKs could interact with ZmCDKA1 directly and affect ZmCDKA1 function, suggesting that C-group ZmICKs are important for cell division regulation.

Identification and characterization of transcription factor ZmEREB94 involved in starch synthesis in maize.

Maize is an important food crop and industrial material owing to its high starch content. However, the mechanism of starch synthesis is not fully elucidated, especially with regard to the expression and regulation of starch synthetic genes. The APETALA2/Ethylene Responsive Factor (AP2/ERF) family plays a crucial role in various biological processes via regulating gene expression. In this study, the ZmEREB94 gene was identified through co-expression analysis. Bioinformatics analysis confirmed that ZmEREB94 belongs to the AP2/ERF family. Expression pattern analysis showed that this protein is strongly expressed in the maize endosperm. A ZmEREB94-GFP fusion protein was localized in the nuclei of onion epidermal cells, and ZmEREB94 showed strong transcriptional activation activity, which indicated that this protein is a transcription factor. In addition, yeast-one hybrid assays and transient expression in maize endosperm showed that ZmEREB94 could directly bind to the ZmSSI promoter and indirectly regulate ZmSh2 and ZmGBSSI expression. Our results revealed that ZmEREB94 might act as a key regulator of starch synthesis in maize.

ZmMYB14 is an important transcription factor involved in the regulation of the activity of the ZmBT1 promoter in starch biosynthesis in maize.

The biosynthesis of starch is a complex process that depends on the regulatory mechanisms of different functional enzymes, and transcriptional regulation plays an important role in this process. Brittle 1, encoded by BT1, is a transporter of adenosine diphosphate-glucose, which plays an important role in the biosynthesis of starch in the endosperm of cereals. Here, we report that the promoter (pZmBT1) of the maize BT1 homolog, ZmBT1, contains an MBSI site (TAACTG), which is important for its activity. Moreover, high expression level of the gene for ZmMYB14 transcription factor was observed in the maize endosperm; its expression pattern was similar to those of the starch synthesis-related genes in maize seeds. ZmMYB14 is a typical 2R-MYB transcription factor localized in the nucleus and possessed transcriptional activation activity. ZmMYB14 could bind to the region of pZmBT1 from -280 to -151 bp and promote its activity through the TAACTG site. It was also observed to promote the activity of pZmSh2, pZmBt2, pZmGBSSI, pZmSSI, and pZmSBE1 in the maize endosperm in transient gene overexpression assays. Furthermore, ZmMYB14 was also shown to bind directly to the promoters of six starch-synthesizing genes, ZmGBSSI, ZmSSI, ZmSSIIa, ZmSBE1, ZmISA1, and ZmISA2 in yeast. These findings indicate that ZmMYB14 functions as a key regulator of ZmBT1 and is closely related to the biosynthesis of starch. Our results provide crucial information related to the regulation of starch biosynthesis in maize and would be helpful in devising strategies for modulating starch production in maize endosperm.

Genetic Analysis in Maize Foundation Parents with Mapping Population and Testcross Population: Ye478 Carried More Favorable Alleles and Using QTL Information Could Improve Foundation Parents.

The development of maize foundation parents is an important part of genetics and breeding research, and applying new genetic information to produce foundation parents has been challenging. In this study, we focused on quantitative trait loci (QTLs) and general combining ability (GCA) of Ye478, a widely used foundation parent in China. We developed three sets of populations for QTL mapping and to analyze the GCA for some agronomic traits. The assessment of 15 traits resulted in the detection of 251 QTLs in six tested environments, with 119 QTLs identified through a joint analysis across all environments. Further, analyses revealed that most favorable alleles for plant type-related traits were from Ye478, and more than half of the favorable alleles for yield-related traits were from R08, another foundation parent used in southwestern China, suggesting that different types of foundation parents carried different favorable alleles. We observed that the GCA for most traits (e.g., plant height and 100-kernel weight) was maintained in the inbred lines descended from the foundation parents. Additionally, the continuous improvement in the GCA of the descendants of the foundation parents was consistent with the main trend in maize breeding programs. We identified three significant genomic regions that were highly conserved in three Ye478 descendants, including the stable QTL for plant height. The GCA for the traits in the F7 generation revealed that the QTLs for the given traits per se were affected by additive effects in the same way in different populations.

Sucrose and ABA regulate starch biosynthesis in maize through a novel transcription factor, ZmEREB156.

Sucrose is not only the carbon source for starch synthesis, but also a signal molecule. Alone or in coordination with ABA, it can regulate the expression of genes involved in starch synthesis. To investigate the molecular mechanisms underlying this effect, maize endosperms were collected from Zea mays L. B73 inbred line 10 d after pollination and treated with sucrose, ABA, or sucrose plus ABA at 28 °C in the dark for 24 h. RNA-sequence analysis of the maize endosperm transcriptome revealed 47 candidate transcription factors among the differentially expressed genes. We therefore speculate that starch synthetic gene expression is regulated by transcription factors induced by the combination of sucrose and ABA. ZmEREB156, a candidate transcription factor, is induced by sucrose plus ABA and is involved in starch biosynthesis. The ZmEREB156-GFP-fused protein was localized in the nuclei of onion epidermal cells, and ZmEREB156 protein possessed strong transcriptional activation activity. Promoter activity of the starch-related genes Zmsh2 and ZmSSIIIa increased after overexpression of ZmEREB156 in maize endosperm. ZmEREB156 could bind to the ZmSSIIIa promoter but not the Zmsh2 promoter in a yeast one-hybrid system. Thus, ZmEREB156 positively modulates starch biosynthetic gene ZmSSIIIa via the synergistic effect of sucrose and ABA.