The m6 A reader SiYTH1 enhances drought tolerance by affecting the messenger RNA stability of genes related to stomatal closure and reactive oxygen species scavenging in Setaria italica.

Foxtail millet (Setaria italica), a vital drought-resistant crop, plays a significant role in ensuring food and nutritional security. However, its drought resistance mechanism is not fully understood. N6 -methyladenosine (m6 A) modification of RNA, a prevalent epi-transcriptomic modification in eukaryotes, provides a binding site for m6 A readers and affects plant growth and stress responses by regulating RNA metabolism. In this study, we unveiled that the YT521-B homology (YTH) family gene SiYTH1 positively regulated the drought tolerance of foxtail millet. Notably, the siyth1 mutant exhibited reduced stomatal closure and augmented accumulation of excessive H2 O2 under drought stress. Further investigations demonstrated that SiYTH1 positively regulated the transcripts harboring m6 A modification related to stomatal closure and reactive oxygen species (ROS) scavenging under drought stress. SiYTH1 was uniformly distributed in the cytoplasm of SiYTH1-GFP transgenic foxtail millet. It formed dynamic liquid-like SiYTH1 cytosol condensates in response to drought stress. Moreover, the cytoplasmic protein SiYTH1 was identified as a distinct m6 A reader, facilitating the stabilization of its directly bound SiARDP and ROS scavenging-related transcripts under drought stress. Furthermore, natural variation analysis revealed SiYTH1AGTG as the dominant allele responsible for drought tolerance in foxtail millet. Collectively, this study provides novel insights into the intricate mechanism of m6 A reader-mediated drought tolerance and presents a valuable genetic resource for improving drought tolerance in foxtail millet breeding.

The NAC transcription factor ZmNAC132 regulates leaf senescence and male fertility in maize.

Leaf senescence is an integral step in the final stages of plant development, as nutrient remobilization from leaves to sink organs is accomplished during this process. NACs compose a large superfamily of plant-specific TFs involved in multiple plant development processes. Here, we identified a maize NAC TF, ZmNAC132, involved in leaf senescence and male fertility. ZmNAC132 expression was tightly linked to leaf senescence in an age-dependent manner. Knockout of ZmNAC132 led to delays in chlorophyll degradation and leaf senescence, whereas overexpression of ZmNAC132 had the opposite effects. ZmNAC132 could bind to and transactivate the promoter of ZmNYE1, a major chlorophyll catabolic gene, to accelerate chlorophyll degradation during leaf senescence. Moreover, ZmNAC132 affected male fertility through the upregulation of ZmEXPB1, an expansin-encoding gene associated with sexual reproduction and other related genes. Together, the results show that ZmNAC132 participates in the regulation of leaf senescence and male fertility through the targeting of different downstream genes in maize.

Maize WRKY28 interacts with the DELLA protein D8 to affect skotomorphogenesis and participates in the regulation of shade avoidance and plant architecture.

Competition for light from neighboring vegetation can trigger the shade-avoidance response (SAR) in plants, which is detrimental to their yield. The molecular mechanisms regulating SAR are well established in Arabidopsis, and some regulators of skotomorphogenesis have been found to be involved in the regulation of the SAR and plant architecture. However, the role of WRKY transcription factors in this process has rarely been reported, especially in maize (Zea mays). Here, we report that maize Zmwrky28 mutants exhibit shorter mesocotyls in etiolated seedlings. Molecular and biochemical analyses demonstrate that ZmWRKY28 directly binds to the promoter regions of the Small Auxin Up RNA (SAUR) gene ZmSAUR54 and the Phytochrome-Interacting Factor (PIF) gene ZmPIF4.1 to activate their expression. In addition, the maize DELLA protein Dwarf Plant8 (D8) interacts with ZmWRKY28 in the nucleus to inhibit its transcriptional activation activity. We also show that ZmWRKY28 participates in the regulation of the SAR, plant height, and leaf rolling and erectness in maize. Taken together, our results reveal that ZmWRKY28 is involved in GA-mediated skotomorphogenic development and can be used as a potential target to regulate SAR for breeding of high-density-tolerant cultivars.

ZmAdSS1 encodes adenylosuccinate synthetase and plays a critical role in maize seed development and the accumulation of nutrients.

Adenylosuccinate synthetase (AdSS, EC.6.3.4.4) is a key enzyme in the de novo synthesis of purine nucleotides in organisms. Its downstream product AMP plays a critical role in the process of energy metabolism, which can affect the content of ADP and ATP. However, impacts of its loss-of-function on plant metabolism and development has been relatively poorly reported. Here, we report the identification and analysis of a maize yu18 mutant obtained by mutagenesis with ethylmethane sulfonate (EMS). The yu18 is a lethal-seed mutant. Map-based cloning and allelic testing confirmed that yu18 encodes adenylosuccinate synthetase and was named ZmAdSS1. ZmAdSS1 is constitutively expressed. In the yu18 mutant, the activity of the ZmAdSS1 enzyme was decreased, which caused AMP content reduced 33.62%. The yu18 mutation significantly suppressed endoreduplication and disrupted nutrient accumulation, resulting in lower starch and protein contents that are responsible for seed filling. Further transcriptome and metabolome analysis revealed dramatic alterations in the carbohydrate metabolic pathway and amino acid metabolic pathway in yu18 kernels. Our findings demonstrate that ZmAdSS1 participates in the synthesis of AMP and affects endosperm development and nutrient accumulation in maize seeds.

SimiR396d targets SiGRF1 to regulate drought tolerance and root growth in foxtail millet.

MicroRNAs play critical roles in growth, development and abiotic stress responses. SimR396d is a miRNA whose expression level is much higher in foxtail millet roots than other tissues. Whether SimR396d is involved in foxtail millet root growth and response to abiotic stress is still unknown. Here, we demonstrate that SimiR396d modulates both drought response and root growth in foxtail millet. The expression of SimiR396d is induced by PEG treatment. Overexpression of SimiR396d enhances drought tolerance and root length, while knockdown SimiR396d expression using target mimics of SimiR396d (MIM396) resulted in reduced drought tolerance and shortened root length. Furthermore, we identified and confirmed a plant-specific transcription factor, growth-regulating factor 1 (SiGRF1), as a direct target of SimiR396d. Overexpression of SiGRF1 in foxtail millet resulted in suppressed root growth and reduced sensitivity to drought stress. Moreover, ethylene signaling is necessary for SimiR396d and SiGRF1 to participate in the regulation of plant root growth. These results revealed a pivotal role of SimiR396d in drought tolerance and root growth in foxtail millet. SimiR396d-SiGRF1 regulatory module provides a strategy to improve drought-stress resistance of crop.

ZmEREB46, a maize ortholog of Arabidopsis WAX INDUCER1/SHINE1, is involved in the biosynthesis of leaf epicuticular very-long-chain waxes and drought tolerance.

The aerial surfaces of plants are covered by a layer of cuticular wax that is composed of long-chain hydrocarbon compounds for protection against adverse environmental conditions. The current study identified a maize (Zea mays L.) APETALA2/ethylene-responsive element-binding protein (AP2/EREBP)-type transcription factor, ZmEREB46. Ectopic expression of ZmEREB46 in Arabidopsis increased the accumulation of epicuticular wax on the leaves and enhanced the drought tolerance of plants. The amounts of C24/C32 fatty acids, C32/C34 aldehydes, C32/C34 1-alcohols and C31 alkanes in zmereb46 (ZmEREB46 knockout mutant) leaves were reduced. The amount of leaf total epicuticular wax decreased approximately 50% in zmereb46. Compared to wild-type LH244 leaves, the cuticle permeability of zmereb46 leaves was increased, which resulted from decreased epicuticular wax load and a thinner cuticle layer. ZmEREB46 had transcriptional activation activity and directly bound to promoter regions of ZmCER2, ZmCER3.2 and ZmKCS1. The zmereb46 seedlings also exhibited reduced drought tolerance. These results, and the observations in ZmEREB46-overexpressing lines, suggest that ZmEREB46 is involved in cuticular metabolism by influencing the biosynthesis of very-long-chain waxes and participates in the cutin biosynthesis pathway. These results are helpful to further analyze the regulatory network of wax accumulation in maize.

Maize GSK3-like kinase ZmSK2 is involved in embryonic development.

Grain size and weight are closely related to the yield of cereal crops. Abnormal development of the embryo, an important part of the grain, not only affects crop yield but also impacts next-generation survival. Here, we found that maize GSK3-like kinase ZmSK2, a homolog of BIN2 in Arabidopsis, is involved in embryonic development. ZmSK2 overexpression resulted in severe BR defective phenotypes and arrested embryonic development at the transition stage, while the zmsk2 knockout lines showed enlarged embryos. ZmSK2 interacts with Aux/IAA-transcription factor 28 (ZmIAA28), a negative regulator of auxin signaling, and the interaction region is the auxin degron "GWPPV" motif of ZmIAA28 domain II. Coexpression of ZmSK2 with ZmIAA28 increased the accumulation of ZmIAA28 in maize protoplasts, which may have been due to phosphorylation by ZmSK2. In conclusion, this study reveals the function of ZmSK2 in maize embryonic development and proposes that ZmSK2-ZmIAA28 may be another link in the signaling pathway that integrates BR and auxin.

Time-series transcriptomics reveals a drought-responsive temporal network and crosstalk between drought stress and the circadian clock in foxtail millet.

Drought stress is a serious factor affecting crop growth and production worldwide. The circadian clock has been identified as key to improving regional adaptability of plants. However, our understanding of the contribution of the circadian clock to drought response and the impacts of drought stress on the circadian clock in plants is still limited. To explore the interactions between the circadian clock and drought stress, foxtail millet seedlings were treated with simulated drought (20% polyethylene glycol-6000) treatment starting at the day (DD) onset zeitgeber time 0 (ZT0, lights on) and at the night (DN) onset zeitgeber time 16 (ZT16, lights off). A high temporal-resolution transcriptomic investigation was performed using DD and DN samples collected at intervals of 2 or 4 h within a 24-h drought-treatment period. Overall, we identified 13 294 drought-responsive genes (DRGs). Among these DRGs, 7931 were common between DD and DN samples, 2638 were specific to DD, and 2725 were specific to DN. Additionally, we identified 1257 circadian genes, of which 67% were DRGs. Interestingly, with drought treatment starting at the day for 8, 12 or 16 h, the circadian phase shifted to 12 h. We also found that the circadian clock led to different day and night drought-responsive pathways. The identification of DRG_Clock (DRG and circadian clock) and DRG_NonClock (DRG and not circadian clock) genes provides a reference for selecting candidate drought resistance genes. Our work reveals the temporal drought-response process and crosstalk between drought stress and the circadian clock in foxtail millet.

ZmGLR, a cell membrane localized microtubule-associated protein, mediated leaf morphogenesis in maize.

Microtubule arrays play notable roles in cell division, cell movement, cell morphogenesis and signal transduction. Due to their important regulation of microtubule dynamic instability and array-ordering processes, microtubule-associated proteins have been a cutting-edge issue in research. Here, a new maize microtubule-associated protein, ZmGLR (Zea mays glutamic acid- and lysine-rich), was found. ZmGLR bundles microtubules in vitro and targets the cell membrane through an interaction between 24 conserved N-terminal amino acids and specific phosphatidylinositol phosphates (PtdInsPs). Increased Ca2+ levels in the cytoplasm lead to ZmGLR partially dissociating from the cell membrane and moving into the cytoplasm to associate with microtubule. Overexpression and RNAi of ZmGLR both resulted in misoriented microtubule arrays, which led to dwarf maize plants and curved leaves. In addition, the expression of ZmGLR was regulated by BR and auxin through ZmBES1 and ZmARF9, respectively. This study reveals that the microtubule-associated protein ZmGLR plays a crucial role in cortical microtubule reorientation and maize leaf morphogenesis.

A DREB-Like Transcription Factor From Maize (Zea mays), ZmDREB4.1, Plays a Negative Role in Plant Growth and Development.

The DREB (dehydration-responsive element binding)-type transcription factors are classified into six subgroups, named A-1 to A-6. The members of DREB A-1 and A-2 subgroups have been reported to be involved in response to various abiotic stresses. However, there were only a few genes belonging to A-3 to A-6 subgroups to be reported. In this study, we cloned a DREB A-4 subgroup gene from maize (Zea mays), ZmDREB4.1, and analyzed its characteristics and functions. ZmDREB4.1 was expressed in roots, stems, and leaves at very low levels. It was not induced by any biotic or abiotic treatment. ZmDREB4.1 was located in the nucleus, could directly bind to the DRE element and functioned as a transcriptional activator. The constitutive expression of ZmDREB4.1 in tobacco (Nicotiana tabacum L.) repressed leaf extension and hypocotyl, petiole and stem elongation. In maize, overexpression of ZmDREB4.1 repressed calli growth and regeneration. Further analysis showed that the smaller leaves of transgenic tobacco resulted from inhibition of cell division. The contents of cytokinin and auxin in transgenic leaves were severely decreased. The shorter hypocotyls, stems and petioles of transgenic tobacco were caused by inhibition of cell elongation. The transgenic hypocotyls, stems and petioles contained reduced gibberellin levels. Application of exogenous GA3 rescued the shorter hypocotyls, stems and petioles, but not the smaller leaves. These results demonstrated that ZmDREB4.1 plays an important role in the negative regulation of plant growth and development.

Functional Analysis of Maize Silk-Specific ZmbZIP25 Promoter.

ZmbZIP25 (Zea mays bZIP (basic leucine zipper) transcription factor 25) is a function-unknown protein that belongs to the D group of the bZIP transcription factor family. RNA-seq data showed that the expression of ZmbZIP25 was tissue-specific in maize silks, and this specificity was confirmed by RT-PCR (reverse transcription-polymerase chain reaction). In situ RNA hybridization showed that ZmbZIP25 was expressed exclusively in the xylem of maize silks. A 5' RACE (rapid amplification of cDNA ends) assay identified an adenine residue as the transcription start site of the ZmbZIP25 gene. To characterize this silk-specific promoter, we isolated and analyzed a 2450 bp (from -2083 to +367) and a 2600 bp sequence of ZmbZIP25 (from -2083 to +517, the transcription start site was denoted +1). Stable expression assays in Arabidopsis showed that the expression of the reporter gene GUS driven by the 2450 bp ZmbZIP25 5'-flanking fragment occurred exclusively in the papillae of Arabidopsis stigmas. Furthermore, transient expression assays in maize indicated that GUS and GFP expression driven by the 2450 bp ZmbZIP25 5'-flanking sequences occurred only in maize silks and not in other tissues. However, no GUS or GFP expression was driven by the 2600 bp ZmbZIP25 5'-flanking sequences in either stable or transient expression assays. A series of deletion analyses of the 2450 bp ZmbZIP25 5'-flanking sequence was performed in transgenic Arabidopsis plants, and probable elements prediction analysis revealed the possible presence of negative regulatory elements within the 161 bp region from -1117 to -957 that were responsible for the specificity of the ZmbZIP25 5'-flanking sequence.

ZmNST3 and ZmNST4 are master switches for secondary wall deposition in maize (Zea mays L.).

Secondary walls are the most abundant biomass produced by plants, and they consist mainly of lignin, cellulose and hemicellulose. Understanding how secondary wall biosynthesis is regulated could potentially provide genetic tools for engineering biomass components, especially in maize and Sorghum bicolor. Although many works have focused on secondary wall biosynthesis in dicotyledons, little has been reported for these monocotyledons. In this study, we cloned two NAC transcriptional factor genes, ZmNST3 and ZmNST4, and analyzed their functions in maize secondary wall formation process. ZmNST3 and ZmNST4 were expressed specifically in secondary wall-forming cells, expression of ZmNST3/4 can restore the pendent phenotype of Arabidopsis nst1nst3 double mutant. ZmNST3/4-overexpressing Arabidopsis and maize displayed a thickened secondary wall in the stem, and knockdown maize showed defective secondary wall deposition. ZmNST3/4 could regulate the expression of ZmMYB109/128/149. Our results revealed that ZmNST3/4 are master switches of the maize secondary wall biosynthesis process and provides new evidence that the secondary wall regulatory pathway is conserved in different plant species.

ZmDof30 Negatively Regulates the Promoter Activity of the Pollen-Specific Gene Zm908.

The maize (Zea mays) pollen-predominant gene Zm908, a novel small-peptide gene, was reported to play critical roles in pollen germination and pollen tube growth in our previous work. In this study, we aimed to explore the regulatory mechanism of Zm908. The putative promoter of Zm908 was cloned and analyzed. The activity analysis of a series of promoter truncations in different tissues of transgenic tobacco plants indicated that the Zm908 promoter is pollen-specific and that the -126 to -68 region is crucial for pollen expression. The 5' deletion analysis of the -126 to -68 region revealed that the -126 to -102 region functions as a transcriptional suppression element. ZmDof30, which is predominantly expressed in pollen and whole anthers, was cloned and characterized. ZmDof30-GFP localized to the nuclei of maize protoplasts and possessed no transcriptional activation activity in a yeast system. ZmDof30 could bind to the AAAG elements in p184 sequence containing the -126 to +58 region of the Zm908 promoter in vitro and in vivo, and negatively regulated p184 activity in tobacco leaves. Collectively, ZmDof30 may function as a Zm908 transcriptional repressor in pollen, and these results may provide a better understanding of the regulation of the Zm908 gene. Additionally, the pollen-specific Zm908 promoter may be valuable for genetically engineering male sterility.

The 5' untranslated region of potato SBgLR gene contributes to pollen-specific expression.

MAIN CONCLUSION: The 5'UTR of SBgLR enhances gene expression by regulating both its transcription and translation. SBgLR (Solanum tuberosum genomic lysine rich) is a pollen-specific gene in Solanum tuberosum that encodes a microtubule-associated protein. The region from -85 to +180 (transcription start site at +1) was determined to be critical for specific expression in pollen grains. Transient and stable expression assays showed that the 5'UTR (from +1 to +184) enhanced gene expression in all detected tissues of transgenic tobacco. Deletion analysis demonstrated that the secondary structure of the 5'UTR had no effect on pollen-specific SBgLR expression, while the region from +31 to +60 was crucial. Further investigation indicated that mRNA expression was slightly decreased when the +31 to +60 region was deleted, but the mRNA decay rate remained unchanged. Mutation analysis also confirmed that the pollen-specific element TTTCT, located at +37, played an important role in pollen-specific expression. Using yeast one-hybrid screening, we isolated a DNA-binding with one finger (Dof) protein gene (StDof23) and an AT-hook motif nuclear-localized (AHL) protein gene (StAHL) from potato pollen. Further investigation indicated that StDof23 interacted with and positively regulated the +31 to +60 region; moreover, StAHL interacted with and negatively regulated the -49 to +60 region. These results demonstrate that the 5'UTR not only enhanced gene expression but also altered the tissue-specific expression pattern by regulating both transcription and translation.

ZmDof3, a maize endosperm-specific Dof protein gene, regulates starch accumulation and aleurone development in maize endosperm.

KEY MESSAGE: To explore the function of Dof transcription factors during kernel development in maize, we first identified Dof genes in the maize genome. We found that ZmDof3 was exclusively expressed in the endosperm of maize kernel and had the features of a Dof transcription factor. Suppression of ZmDof3 resulted in a defective kernel phenotype with reduced starch content and a partially patchy aleurone layer. The expression levels of starch synthesis-related genes and aleurone differentiation-associated genes were down-regulated in ZmDof3 knockdown kernels, indicating that ZmDof3 plays an important role in maize endosperm development. The maize endosperm, occupying a large proportion of the kernel, plays an important role in seed development and germination. Current knowledge regarding the regulation of endosperm development is limited. Dof proteins, a family of plant-specific transcription factors, play critical roles in diverse biological processes. In this study, an endosperm-specific Dof protein gene, ZmDof3, was identified in maize through genome-wide screening. Suppression of ZmDof3 resulted in a defective kernel phenotype. The endosperm of ZmDof3 knockdown kernels was loosely packed with irregular starch granules observed by electronic microscope. Through genome-wide expression profiling, we found that down-regulated genes were enriched in GO terms related to carbohydrate metabolism. Moreover, ZmDof3 could bind to the Dof core element in the promoter of starch biosynthesis genes Du1 and Su2 in vitro and in vivo. In addition, the aleurone at local position in mature ZmDof3 knockdown kernels varied from one to three layers, which consisted of smaller and irregular cells. Further analyses showed that knockdown of ZmDof3 reduced the expression of Nkd1, which is involved in aleurone cell differentiation, and that ZmDof3 could bind to the Dof core element in the Nkd1 promoter. Our study reveals that ZmDof3 functions in maize endosperm development as a positive regulator in the signaling system controlling starch accumulation and aleurone development.

A Non-specific Setaria italica Lipid Transfer Protein Gene Plays a Critical Role under Abiotic Stress.

Lipid transfer proteins (LTPs) are a class of cysteine-rich soluble proteins having small molecular weights. LTPs participate in flower and seed development, cuticular wax deposition, also play important roles in pathogen and abiotic stress responses. A non-specific LTP gene (SiLTP) was isolated from a foxtail millet (Setaria italica) suppression subtractive hybridization library enriched for differentially expressed genes after abiotic stress treatments. A semi-quantitative reverse transcriptase PCR analysis showed that SiLTP was expressed in all foxtail millet tissues. Additionally, the SiLTP promoter drove GUS expression in root tips, stems, leaves, flowers, and siliques of transgenic Arabidopsis. Quantitative real-time PCR indicated that the SiLTP expression was induced by NaCl, polyethylene glycol, and abscisic acid (ABA). SiLTP was localized in the cytoplasm of tobacco leaf epidermal cells and maize protoplasts. The ectopic expression of SiLTP in tobacco resulted in higher levels of salt and drought tolerance than in the wild type (WT). To further assess the function of SiLTP, SiLTP overexpression (OE) and RNA interference (RNAi)-based transgenic foxtail millet were obtained. SiLTP-OE lines performed better under salt and drought stresses compared with WT plants. In contrast, the RNAi lines were much more sensitive to salt and drought compared than WT. Electrophoretic mobility shift assays and yeast one-hybrids indicated that the transcription factor ABA-responsive DRE-binding protein (SiARDP) could bind to the dehydration-responsive element of SiLTP promoter in vitro and in vivo, respectively. Moreover, the SiLTP expression levels were higher in SiARDP-OE plants compared than the WT. These results confirmed that SiLTP plays important roles in improving salt and drought stress tolerance of foxtail millet, and may partly be upregulated by SiARDP. SiLTP may provide an effective genetic resource for molecular breeding in crops to enhance salt and drought tolerance levels.

Genome-Wide Analysis of the Lysine Biosynthesis Pathway Network during Maize Seed Development.

Lysine is one of the most limiting essential amino acids for humans and livestock. The nutritional value of maize (Zea mays L.) is reduced by its poor lysine content. To better understand the lysine biosynthesis pathway in maize seed, we conducted a genome-wide analysis of the genes involved in lysine biosynthesis. We identified lysine biosynthesis pathway genes (LBPGs) and investigated whether a diaminopimelate pathway variant exists in maize. We analyzed two genes encoding the key enzyme dihydrodipicolinate synthase, and determined that they contribute differently to lysine synthesis during maize seed development. A coexpression network of LBPGs was constructed using RNA-sequencing data from 21 developmental stages of B73 maize seed. We found a large set of genes encoding ribosomal proteins, elongation factors and zein proteins that were coexpressed with LBPGs. The coexpressed genes were enriched in cellular metabolism terms and protein related terms. A phylogenetic analysis of the LBPGs from different plant species revealed different relationships. Additionally, six transcription factor (TF) families containing 13 TFs were identified as the Hub TFs of the LBPGs modules. Several expression quantitative trait loci of LBPGs were also identified. Our results should help to elucidate the lysine biosynthesis pathway network in maize seed.

SiASR4, the Target Gene of SiARDP from Setaria italica, Improves Abiotic Stress Adaption in Plants.

Drought and other types of abiotic stresses negatively affect plant growth and crop yields. The abscisic acid-, stress-, and ripening-induced (ASR) proteins play important roles in the protection of plants against abiotic stress. However, the regulatory pathway of the gene encoding this protein remains to be elucidated. In this study, the foxtail millet (Setaria italica) ASR gene, SiASR4, was cloned and characterized. SiASR4 localized to the cell nucleus, cytoplasm and cytomembrane, and the protein contained 102 amino acids, including an ABA/WDS (abscisic acid/water-deficit stress) domain, with a molecular mass of 11.5 kDa. The abundance of SiASR4 transcripts increased after treatment with ABA, NaCl, and PEG in foxtail millet seedlings. It has been reported that the S. italica ABA-responsive DRE-binding protein (SiARDP) binds to a DNA sequence with a CCGAC core and that there are five dehydration-responsive element (DRE) motifs within the SiASR4 promoter. Our analyses demonstrated that the SiARDP protein could bind to the SiASR4 promoter in vitro and in vivo. The expression of SiASR4 increased in SiARDP-overexpressing plants. SiASR4-transgenic Arabidopsis and SiASR4-overexpressing foxtail millet exhibited enhanced tolerance to drought and salt stress. Furthermore, the transcription of stress-responsive and reactive oxygen species (ROS) scavenger-associated genes was activated in SiASR4 transgenic plants. Together, these findings show that SiASR4 functions in the adaption to drought and salt stress and is regulated by SiARDP via an ABA-dependent pathway.

Microtubule-Associated Protein SBgLR Facilitates Storage Protein Deposition and Its Expression Leads to Lysine Content Increase in Transgenic Maize Endosperm.

Maize (Zea mays) seed is deficient in protein and lysine content. Many studies have been made to improve the nutritional quality of maize seeds. Previously, we reported the role of a natural lysine-rich protein gene SBgLR in increasing protein and lysine content. However, how the SBgLR improves lysine and protein content remains unclear. Here, the reasons and possible mechanism for SBgLR in protein and lysine improvement have been analyzed and discussed. Through seed-specific expression of SBgLR, we obtained transgenic maize with the simultaneously increased lysine and protein contents. High-protein and high-lysine characters were stably inherited across generations. The expression of SBgLR in maize kernels increased the accumulation of both zeins and non-zein proteins. Transmission electron microscopy showed that the number of protein bodies (PBs) was increased obviously in SBgLR transgenic immature endosperms with the morphology and structure of PBs unchanged. The proteinaceous matrix was more abundant in transgenic mature endosperms under scanning electron microscopy. The stabilities of zein and lysine-rich non-zein genes were also increased in transgenic endosperms. Finally, the potential application of SBgLR in maize nutrient improvement was evaluated. This study shows that a cytoskeleton-associated protein has potential applicable value in crop nutrient improving, and provided a feasible strategy for improvement of maize grain quality.

Seed-Specific Expression of the Arabidopsis AtMAP18 Gene Increases both Lysine and Total Protein Content in Maize.

Lysine is the most limiting essential amino acid for animal nutrition in maize grains. Expression of naturally lysine-rich protein genes can increase the lysine and protein contents in maize seeds. AtMAP18 from Arabidopsis thaliana encoding a microtubule-associated protein with high-lysine content was introduced into the maize genome with the seed-specific promoter F128. The protein and lysine contents of different transgenic offspring were increased prominently in the six continuous generations investigated. Expression of AtMAP18 increased both zein and non-zein protein in the transgenic endosperm. Compared with the wild type, more protein bodies were observed in the endosperm of transgenic maize. These results implied that, as a cytoskeleton binding protein, AtMAP18 facilitated the formation of protein bodies, which led to accumulation of both zein and non-zein proteins in the transgenic maize grains. Furthermore, F1 hybrid lines with high lysine, high protein and excellent agronomic traits were obtained by hybridizing T6 transgenic offspring with other wild type inbred lines. This article provides evidence supporting the use of cytoskeleton-associated proteins to improve the nutritional value of maize.