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Maize is a staple food, feed, and industrial crop. One of the major stresses on maize production is heat stress, which is usually accompanied by other stresses, such as drought or salinity. In this review, we compared the effects of high temperatures on maize production in China. Heat stress disturbs cellular homeostasis and impedes growth and development in plants. Plants have evolved a variety of responses to minimize the damage related to high temperatures. This review summarized the responses in different cell organelles at elevated temperatures, including transcriptional regulation control in the nuclei, unfolded protein response and endoplasmic reticulum-associated protein quality control in the endoplasmic reticulum (ER), photosynthesis in the chloroplast, and other cell activities. Cells coordinate their activities to mediate the collective stresses of unfavorable environments. Accordingly, we evaluated heat stress at the local and systemic levels in in maize. We discussed the physiological and morphological changes in sensing tissues in response to heat stress in maize and the existing knowledge on systemically acquired acclimation in plants. Finally, we discussed the challenges and prospects of promoting corn thermotolerance by breeding and genetic manipulation.

ZmPTF1 regulates drought tolerance in maize by promoting root development and ABA synthesis, by binding to the G-box in the promoter and activating the expression of NCEDs, CBF4, NAC081, and NAC30., Drought stress is the most important environmental stress limiting maize production. ZmPTF1, a phosphate starvation-induced basic helix-loop-helix (bHLH) transcription factor, contributes to root development and low-phosphate tolerance in maize. Here, ZmPTF1 expression, drought tolerance, and the underlying mechanisms were studied by using maize ZmPTF1 overexpression lines and mutants. ZmPTF1 was found to be a positive regulator of root development, ABA synthesis, signalling pathways, and drought tolerance. ZmPTF1 was also found to bind to the G-box element within the promoter of 9-cis-epoxycarotenoid dioxygenase (NCED), C-repeat-binding factor (CBF4), ATAF2/NAC081, NAC30, and other transcription factors, and to act as a positive regulator of the expression of those genes. The dramatically upregulated NCEDs led to increased abscisic acid (ABA) synthesis and activation of the ABA signalling pathway. The up-regulated transcription factors hierarchically regulate the expression of genes involved in root development, stress responses, and modifications of transcriptional regulation. The improved root system, increased ABA content, and activated ABA-, CBF4-, ATAF2-, and NAC30-mediated stress responses increased the drought tolerance of the ZmPTF1 overexpression lines, while the mutants showed opposite trends. This study describes a useful gene for transgenic breeding and helps us understand the role of a bHLH protein in plant root development and stress responses.

Salinity is a major factor limiting plant growth and agricultural production worldwide. Glycine betaine (GB) is one of the most universal osmoprotectants that protects plants from environmental stresses. In this study, transgenic cotton co-expressing ApGSMT2g and ApDMT2g was generated by Agrobacterium-mediated transformation. Compared with wild-type (WT), co-expression of ApGSMT2g and ApDMT2g in cotton results in higher GB amounts, higher relative water content (RWC), lower osmotic potential, more K+, and less Na+ under salt stress, which contributes to maintaining intracellular osmoregulation and K+/Na+ homeostasis and thus confers higher salt tolerance and more vigorous growth. Furthermore, co-expressing ApGSMT2g and ApDMT2g in cotton leads to better performance of PSII, greater photosynthesis capacity, and finally, improves plant growth and increases cotton seed yield compared to WT under salt stress. The reason for the better performance of PSII in transgenic cotton is the higher quantum yield and a more reasonable quantum ratio distribution than WT under salt stress. Co-expressing ApGSMT2g and ApDMT2g in cotton also reduces membrane damage and increases superoxide dismutase (SOD) activity compared to WT under salt stress. Our results indicate that transgenic ApGSMT2g and ApDMT2g cotton shows higher salt tolerance and more seed cotton yield in saline fields compared to wild-type.

In plants, bZIP (basic leucine zipper) transcription factors regulate diverse processes such as development and stress responses. However, few of these transcription factors have been functionally characterized in maize (Zea mays). In this study, we characterized the bZIP transcription factor gene ZmbZIP4 from maize. ZmbZIP4 was differentially expressed in various organs of maize and was induced by high salinity, drought, heat, cold, and abscisic acid treatment in seedlings. A transactivation assay in yeast demonstrated that ZmbZIP4 functioned as a transcriptional activator. A genome-wide screen for ZmbZIP4 targets by immunoprecipitation sequencing revealed that ZmbZIP4 could positively regulate a number of stress response genes, such as ZmLEA2, ZmRD20, ZmRD21, ZmRab18, ZmNHX3, ZmGEA6, and ZmERD, and some abscisic acid synthesis-related genes, including NCED, ABA1, AAO3, and LOS5. In addition, ZmbZIP4 targets some root development-related genes, including ZmLRP1, ZmSCR, ZmIAA8, ZmIAA14, ZmARF2, and ZmARF3, and overexpression of ZmbZIP4 resulted in an increased number of lateral roots, longer primary roots, and an improved root system. Increased abscisic acid synthesis by overexpression of ZmbZIP4 also can increase the plant’s ability to resist abiotic stress. Thus, ZmbZIP4 is a positive regulator of plant abiotic stress responses and is involved in root development in maize.

NAC proteins constitute one of the largest families of plant-specific transcription factors, and a number of these proteins participate in the regulation of plant development and responses to abiotic stress. T. HALOPHILA STRESS RELATED NAC1 (TsNAC1), cloned from the halophyte Thellungiella halophila, is a NAC transcription factor gene, and its overexpression can improve abiotic stress resistance, especially in salt stress tolerance, in both T. halophila and Arabidopsis (Arabidopsis thaliana) and retard the growth of these plants. In this study, the transcriptional activation activity of TsNAC1 and RD26 from Arabidopsis was compared with the target genes’ promoter regions of TsNAC1 from T. halophila, and the results showed that the transcriptional activation activity of TsNAC1 was higher in tobacco (Nicotiana tabacum) and yeast. The target sequence of the promoter from the target genes also was identified, and TsNAC1 was shown to target the positive regulators of ion transportation, such as T. HALOPHILA H+-PPASE1, and the transcription factors MYB HYPOCOTYL ELONGATION-RELATED and HOMEOBOX12. In addition, TsNAC1 negatively regulates the expansion of cells, inhibits LIGHT-DEPENDENT SHORT HYPOCOTYLS1 and UDP-XYLOSYLTRANSFERASE2, and directly controls the expression of MULTICOPY SUPPRESSOR OF IRA14. Based on these results, we propose that TsNAC1 functions as an important upstream regulator of plant abiotic stress responses and vegetative growth.

Summary Maize is a globally important food, feed crop and raw material for the food and energy industry. Plant architecture optimization plays important roles in maize yield improvement. PIN‐FORMED (PIN) proteins are important for regulating auxin spatiotemporal asymmetric distribution in multiple plant developmental processes. In this study, ZmPIN1a overexpression in maize increased the number of lateral roots and inhibited their elongation, forming a developed root system with longer seminal roots and denser lateral roots. ZmPIN1a overexpression reduced plant height, internode length and ear height. This modification of the maize phenotype increased the yield under high‐density cultivation conditions, and the developed root system improved plant resistance to drought, lodging and a low‐phosphate environment. IAA concentration, transport capacity determination and application of external IAA indicated that ZmPIN1a overexpression led to increased IAA transport from shoot to root. The increase in auxin in the root enabled the plant to allocate more carbohydrates to the roots, enhanced the growth of the root and improved plant resistance to environmental stress. These findings demonstrate that maize plant architecture can be improved by root breeding to create an ideal phenotype for further yield increases.

Background Drought is a serious causal factor of reduced crop yields than any other abiotic stresses. As one of the most widely distributed crops, maize plants frequently suffer from drought stress, which causes great losses in the final kernel yield. Drought stress response in plants showed tissue- and developmental stage-specific characteristics. Results In this study, the ears at the V9 stage, kernels and ear leaf at the 5DAP (days after pollination) stage of maize were used for morphological, physiological and comparative transcriptomics analysis to understand the different features of “sink” or “source” organs and the effects on kernel yield under drought stress conditions. The ABA-, NAC-mediate signaling pathway, osmotic protective substance synthesis and protein folding response were identified as common drought stress response in the three organs. Tissue-specific drought stress responses and the regulators were identified, they were highly correlated with growth, physiological adaptation and yield loss under drought stress. For ears, drought stress inhibited ear elongation, led to the abnormal differentiation of the paired spikelet, and auxin signaling involved in the regulation of cell division and growth and primordium development changes. In the kernels, reduced kernel size caused by drought stress was observed, and the obvious differences of auxin, BR and cytokine signaling transduction appeared, which indicated the modification in carbohydrate metabolism, cell differentiation and growth retardation. For the ear leaf, dramatically and synergistically reduced the expression of photosynthesis genes were observed when suffered from drought stress, the ABA- and NAC- mediate signaling pathway played important roles in the regulation of photosynthesis. Conclusions Transcriptomic changes caused by drought were highly correlated with developmental and physiological adaptation, which was closely related to the final yield of maize, and a sketch of tissue- and developmental stage-specific responses to drought stress in maize was drafted. Electronic supplementary material The online version of this article (10.1186/s12870-019-1941-5) contains supplementary material, which is available to authorized users.

The local auxin gradient has a decisive role in auxin signaling, auxin-mediated development and abiotic stress response. PINFORMED (PIN)-formed auxin efflux transporters are very important for determining the direction of auxin transport and maintaining a local auxin concentration gradient. In this study, all candidate PIN genes from the current maize genome sequence database were identified and categorized based on amino acid similarity. The expression pattern of these PINs was analyzed in maize inbred line DH4866, which was selected from the progeny of 7922 and 478, and served as the female parent line of many hybrids in Shandong Denghai Seeds Co Ltd (China). Tissue-specific expression patterns indicated that they may have different roles in different stages of development, especially in the root system. Promoter motif analysis of four maize PIN1 genes and their expression levels in response to NAA, low phosphate levels and PEG treatment indicated that ZmPIN1a and ZmPIN1b may contribute more than ZmPIN1c and ZmPIN1d to root growth regulation and abiotic stress response. Analysis of the ZmPIN1a and ZmPIN1b transgenic lines (in DH4866) indicated that they have different effects on root development and growth, with ZmPIN1a increasing the number of lateral roots and inhibiting their elongation to form a developed root system, while ZmPIN1b increases root biomass by promoting the growth of both lateral and seminal roots. These results indicated that maize PIN1 genes function in coordination during maize development and in response to abiotic stress.

The emergence and survival of cotton seedlings is crucial for cotton cultivation in saline fields. Limited seed cotton yield in saline fields is also a matter of concern for farmers. The expression of TsVP, a Thellungiella halophila gene encoding a vacuolar proton pumping pyrophosphatase, has been shown to improve the salinity tolerance of transgenic cotton under greenhouse conditions. However, the potential for TsVP to improve the seed cotton yield in saline fields has yet to be evaluated. In this study, the time of emergence, emergence rate, survival rate, carbon assimilation capacity during bud stage, seed cotton yield, and cotton fibre quality were determined in saline field trials using TsVP-overexpressing transgenic cotton and wild-type cotton plants. In a saline field, TsVP-overexpressing transgenic cotton emerged two days earlier than wild-type plants, and displayed greater emergence rate and survival rate. The seed cotton yield of TsVP-overexpressing transgenic cotton plants increased by an average of 14.81 % compared with that of wild-type plants, and cotton fibre quality also improved. In greenhouse conditions, TsVP-overexpressing plants also required a shorter time for 50 % emergence than wild-type plants when the NaCl concentration was greater than 100 mM. This research indicates that TsVP has the potential to improve seed cotton yield in saline fields.

Heterologous expression of a transcription factor (TF) gene in a related species is a useful method for crop breeding and the identification of gene function. The differences in phenotype and target gene expression between HE lines (with the heterologous expression of an ortholog) and OX lines (with an overexpressed native gene) must be understood. EsNAC1, encoding a NAC protein and the ortholog of RD26 in Arabidopsis, was cloned from Eutrema salsugineum and introduced into Arabidopsis. The heterologous expression of EsNAC1 retarded the vegetative growth of Arabidopsis, and the transgenic plants (HE lines) showed much greater resistance to salt and oxidative stress than the wild type, Col-0. The HE lines accumulated 2.8-fold (8-h light) of starch, 1.42-fold of Chlorophyll a and 1.31-fold of Chlorophyll b than Col-0 during the light period, with obvious differences compared to the RD26OX line. A genome-wide ChIP (chromatin immunoprecipitation analysis)-on-chip assay revealed that EsNAC1 targeted promoters of different genes compared to RD26. In HE lines, EsNAC1 could specifically upregulate the expression level of TF genes NAC DOMAIN CONTAINING PROTEIN 62 (ANAC062), INTEGRASE-TYPE DNA-BINDING PROTEIN (TINY2), and MYB HYPOCOTYL ELONGATION-RELATED (MYBH) to show more effective abiotic stress resistance than RD26OX lines. Moreover, DELTA1-PYRROLINE-5-CARBOXYLATE SYNTHASE 1 (P5CS1), TRYPTOPHAN BIOSYNTHESIS 2 (TRP2) or GALACTINOL SYNTHASE 2 (GOLS2), was also specifically regulated by EsNAC1 to retard the vegetative growth of HE lines, but not the brassinosteroid singling pathway in RD26OX lines. These differences in phenotypes and metabolism between the HE lines and the RD26OX line implied that the differential features could be produced from the diversity of target genes in the transgenic plants when the ortholog was introduced.

Phytohormone signaling is involved in the low-phosphate (LP) response and causes root system changes. To understand the roles of auxin and gibberellic acid (GA) in the maize response to LP stress, inbred line Q319 was used to identify the changes in root morphology and the gene expression response to LP stress with or without exogenous auxin, GA or their inhibitors. The root morphology, IAA and GAs concentration and genes related to the LP response, cell elongation and division, auxin transport and signaling, and GA synthesis and signaling were analyzed. The LP-induced maize root morphological adaption was dependent on changes in the expression of related genes, like IPS1, pht1;1 LPR1b, KRPs, and EXPB1-4. The altered local auxin concentration and signaling were involved in promoting axial root elongation and reducing lateral root density and length under LP conditions, which were regulated by PID and PP2A activity and the auxin signaling pathway. The upregulation of the GA synthesis genes AN1, GA20ox1, and GA20ox2 and the downregulation of the GA inactive genes GA2ox1 and GA2ox2 were observed in maize roots subjected to LP stress, and the increased GA biosynthesis and signaling were involved in root growth. Both hormones participate in LP stress response and jointly regulated root modification and LP acclimation in maize.

ZmNF-YB16 is a basic NF-YB superfamily member and a member of a transcription factor complex composed of NF-YA, NF-YB, and NF-YC in maize. ZmNF-YB16 was transformed into the inbred maize line B104 to produce homozygous overexpression lines. ZmNF-YB16 overexpression improves dehydration and drought stress resistance in maize plants during vegetative and reproductive stages by maintaining higher photosynthesis and increases the maize grain yield under normal and drought stress conditions. Based on the examination of differentially expressed genes between the wild-type (WT) and transgenic lines by quantitative real time PCR (qRT-PCR), ZmNF-YB16 overexpression increased the expression of genes encoding antioxidant enzymes, the antioxidant synthase, and molecular chaperones associated with the endoplasmic reticulum (ER) stress response, and improved protection mechanism for photosynthesis system II. Plants that overexpression ZmNF-YB16 showed a higher rate of photosynthesis and antioxidant enzyme activity, better membrane stability and lower electrolyte leakage under control and drought stress conditions. These results suggested that ZmNF-YB16 played an important role in drought resistance in maize by regulating the expression of a number of genes involved in photosynthesis, the cellular antioxidant capacity and the ER stress response.

Phosphate (Pi) limitation is a constraint for plant growth and development in many natural and agricultural ecosystems. In this study, a gene encoding Zea mays L. protein phosphatase 2A regulatory subunit A, designated ZmPP2AA1, was induced in roots by low Pi availability. The function of the ZmPP2AA1 gene in maize was analyzed using overexpression and RNA interference. ZmPP2AA1 modulated root gravitropism, negatively regulated primary root (PR) growth, and stimulated the development of lateral roots (LRs). A detailed characterization of the root system architecture (RSA) in response to different Pi concentrations with or without indole-3-acetic acid and 1-N-naphthylphthalamic acid revealed that auxin was involved in the RSA response to low Pi availability. Overexpression of ZmPP2AA1 enhanced tolerance to Pi starvation in transgenic maize in hydroponic and soil pot experiments. An increased dry weight (DW), root-to-shoot ratio, and total P content and concentration, along with a delayed and reduced accumulation of anthocyanin in overexpressing transgenic maize plants coincided with their highly branched root system and increased Pi uptake capability under low Pi conditions. Inflorescence development of the ZmPP2AA1 overexpressing line was less affected by low Pi stress, resulting in higher grain yield per plant under Pi deprivation. These data reveal the biological function of ZmPP2AA1, provide insights into a linkage between auxin and low Pi responses, and drive new strategies for the efficient utilization of Pi by maize.

Main conclusion RNAi technology was applied to suppress the expression of starch branching enzyme IIa and IIb and to increase amylose content in maize endosperm, and stably inherited high-amylose maize lines were obtained. Amylose is an important material for industries and in the human diet. Maize varieties with endosperm amylose content (AC) of greater than 50 % are termed amylomaize, and possess high industrial application value. The high-amylose trait is controlled by multi-enzyme reaction and intricate gene–environment interaction. Starch branching enzymes are key factors for regulating the branching profiles of starches. In this paper, we report the successful application of RNAi technology for improving amylose content in maize endosperm through the suppression of the ZmSBEIIa and ZmSBEIIb genes by hairpin SBEIIRNAi constructs. These SBEIIRNAi transgenes led to the down-regulation of ZmSBEII expression and SBE activity to various degrees and altered the morphology of starch granules. Transgenic maize lines with AC of up to 55.89 % were produced, which avoided the significant decreases in starch content and grain yield that occur in high-amylose ae mutant. Novel maize lines with high AC offer potential benefits for high-amylose maize breeding. A comparison of gene silencing efficiency among transgenic lines containing different hpSBEIIRNA constructs demonstrated that (1) it was more efficient to use both ZmSBEIIa and ZmSBEIIb specific regions than to use the conserved domain as the inverted repeat arms; (2) the endosperm-specific promoter of the 27-kDa γ-zein provided more efficient inhibition than the CaMV 35S promoter; and (3) inclusion of the catalase intron in the hpSBEIIRNA constructs provided a better silencing effect than the chalcone synthase intron in the hpRNA construct design for suppression of the SBEII subfamily in endosperm.

Summary Grain weight and grain number are important crop yield determinants. DA1 and DAR1 are the ubiquitin receptors that function as the negative regulators of cell proliferation during development in Arabidopsis. An arginine to lysine mutant at amino acid site 358 could lead to the da1-1 phenotype, which results in an increased organ size and larger seeds. In this study, the mutated ZmDA1 (Zmda1) and mutated ZmDAR1 (Zmdar1) driven by the maize ubiquitin promoter was separately introduced into maize elite inbred line DH4866. The grain yield of the transgenic plants was 15% greater than that of the wild type in three years of field trials due to improvements in the grain number, weight and starch content. Interestingly, the over-expression of Zmda1 and Zmdar1 promoted kernel development, resulting in a more developed basal endosperm transfer cell layer (BETL) than WT and enhanced expression of starch synthase genes. This study suggests that the over-expression of the mutated ZmDA1 or ZmDAR1 genes improves the sugar imports into the sink organ and starch synthesis in maize kernels. This article is protected by copyright. All rights reserved.

Phosphatidylinositol (PtdIns) synthase is a key enzyme in the phospholipid pathway and catalyses the formation of PtdIns. PtdIns is not only a structural component of cell membranes, but also the precursor of the phospholipid signal molecules that regulate plant response to environment stresses. Here, we obtained transgenic maize constitutively overexpressing or underexpressing PIS from maize (ZmPIS) under the control of a maize ubiquitin promoter. Transgenic plants were confirmed by PCR, Southern blotting analysis and real-time RT-PCR assay. The electrospray ionization tandem mass spectrometry (ESI-MS/MS)-based lipid profiling analysis showed that, under drought stress conditions, the overexpression of ZmPIS in maize resulted in significantly elevated levels of most phospholipids and galactolipids in leaves compared with those in wild type (WT). At the same time, the expression of some genes involved in the phospholipid metabolism pathway and the abscisic acid (ABA) biosynthesis pathway including ZmPLC, ZmPLD, ZmDGK1, ZmDGK3, ZmPIP5K9, ZmABA1, ZmNCED, ZmAAO1, ZmAAO2 and ZmSCA1 was markedly up-regulated in the overexpression lines after drought stress. Consistent with these results, the drought stress tolerance of the ZmPIS sense transgenic plants was enhanced significantly at the pre-flowering stages compared with WT maize plants. These results imply that ZmPIS regulates the plant response to drought stress through altering membrane lipid composition and increasing ABA synthesis in maize.

Background The low-phosphate-tolerant maize mutant Qi319-96 was obtained from Qi319 through cellular engineering. To elucidate the molecular mechanisms underlying the low-phosphate tolerance of this mutant, we performed comparative proteome analyses of the leaves of Qi319-96 and Qi319 under inorganic phosphate (Pi)-sufficient and Pi-deficient conditions. Results Low-phosphorus levels limit plant growth and metabolism. Although the overall phosphorus contents of shoots were not significantly different between Qi319 and Qi319-96, the Pi level of Qi319-96 was 52.94 % higher than that of Qi319. Under low phosphorus conditions, Qi319-96 had increased chlorophyll levels and enhanced photosynthesis. The changes in starch and sucrose contents under these conditions also differed between genotypes. The proteomic changes included 29 (Pi-sufficient) and 71 (Pi-deficient) differentially expressed proteins involved in numerous metabolic processes. Proteome and physiological analyses revealed that Qi319-96 could better remodel the lipid composition of membranes and had higher V-ATPase activity levels than Qi319 under low-phosphate starvation, which enhanced the recycling of intracellular Pi, as reflected by its increased Pi levels. Chlorophyll biosynthesis was improved and the levels, and activities, of several Calvin cycle and “CO2 pump” enzymes were greater in Qi319-96 than in Qi319, which led to a higher rate of photosynthesis under low-phosphate stress in this line compared with in Qi319. Conclusions Our results suggest that the increased tolerance of the maize mutant Qi319-96 to low-phosphate levels is owing to its ability to increase Pi availability. Additionally, inbred lines of maize with low-P-tolerant traits could be obtained effectively through cellular engineering. Electronic supplementary material The online version of this article (doi:10.1186/s12870-016-0825-1) contains supplementary material, which is available to authorized users.

Glycinebetaine plays an important role in the protection mechanism of many plants under various stress conditions. In this study, genetically engineered maize plants with an enhanced ability to synthesise glycinebetaine (GB) were produced by introducing two genes, glycine sarcosine methyltransferase gene (ApGSMT2) and dimethylglycine methyltransferase gene (ApDMT2), from the bacterium Aphanothece halophytica. Southern blotting and RT-PCR analysis demonstrated that the two genes were integrated into the maize genome and expressed. The increased expression levels of ApGSMT2 and ApDMT2 under drought conditions facilitated GB accumulation in the leaves of transgenic maize plants and conferred improved drought tolerance. Under drought conditions, the transgenic plants showed an increased accumulation of sugars and free amino acids, greater chlorophyll content, a higher photosynthesis rate and biomass, and lower malondialdehyde and electrolyte leakage compared to the wild-type; these results suggest that GB provides vital protection against drought stress. Under normal conditions, the transgenic plants did not show decreased biomass and productivity, which indicated that the co-expression of ApGSMT2 and ApDMT2 in maize plays an important role in its tolerance to drought stress and does not lead to detrimental effects. It was concluded that the co-expression of ApGSMT2 and ApDMT2 in maize is an effective approach to enhancing abiotic stress tolerance in maize breeding programmes.

Maize is a globally important crop, and a low phosphate (LP) supply frequently limits maize yields in many areas. microRNAs (miRNAs) play important roles in plant development and environmental adaptation. In this study, spatio-temporal miRNA transcript profiling and some of the target genes in the roots and leaves of the maize inbred line Q319 were analyzed in response to LP. Complex small RNA populations were detected after LP culture, and they displayed different patterns in the roots and leaves. Differentially expressed miRNAs can be grouped into 'early' miRNAs, which respond rapidly and are often non-specific to phosphate deficiency, and 'late' miRNAs, which alter the morphology, physiology or metabolism of plants upon prolonged phosphate deficiency. miR827 and miR399-mediated posttranscriptional pathway responses to phosphate availability were conserved and species-specific in maize. Abiotic stress-related miRNAs were engaged in interactions with different signaling and/or metabolic pathways. Auxin-related miRNAs and their targets' expression may be involved in root architecture modification and upland growth retardation in maize when subjected to LP. The changes that were found in the expression of miRNAs and their target genes suggested that miRNA regulation/alterations are pivotal mechanisms in maize adaptations to LP environments. A complex regulatory mechanism involving miRNAs in response to the LP environment is present in maize.

Homozygous transgenic cotton (Gossypium hirsutum L.) plants that accumulated glycinebetaine (GB) in larger quantities were more tolerant to salt than wild-type (WT) plants. Four transgenic lines, namely 1, 3, 4, and 5, accumulated significantly higher levels of GB than WT plants did both before and after salt stress. At 175 and 275 mM NaCl, seeds of all the transgenic lines germinated earlier and recorded a higher final germination percentage, and the seedlings grew better, than those of the WT. Under salt stress, all the lines showed some characteristic features of salt tolerance, such as higher leaf relative water content (RWC), higher photosynthesis, better osmotic adjustment (OA), lower percentage of ion leakage, and lower peroxidation of the lipid membrane. Levels of endogenous GB in the transgenic plants were positively correlated with RWC and OA. The results indicate that GB in transgenic cotton plants not only maintains the integrity of cell membranes but also alleviates osmotic stress caused by high salinity. Lastly, the seed cotton yield of transgenic lines 4 and 5 was significantly higher than that of WT plants in saline soil. This research indicates that betA gene has the potential to improve crop’s salt tolerance in areas where salinity is limiting factors for agricultural productivity.

Drought is a major agricultural menace reducing crop productivity and limiting the successful realization of land potential throughout the world. Therefore, breeding common wheat with improved drought-tolerance via genetic manipulation is of great importance. We have introduced the betA gene encoding choline dehydrogenase from Escherichia coli into common wheat (Triticum aestivum L.) by Agrobacterium-mediated transformation. Various levels of expression of the betA gene were confirmed by RT-PCR among the transgenic lines and different levels of glycine betaine accumulation were detected in these lines. Several wheat transgenic lines with different betA expression levels in the T3 generation and wild-type (WT) were selected to test their performance under drought stress conditions. Water deficit in plants caused a reduction in photosynthesis and activity of the PSII complex and resulted in increased accumulation of osmolytes. Drought stress also led to lower membrane stability along with much higher activities of superoxide dismutase and peroxidase in all wheat lines. However, wheat lines that were transgenic for the betA gene were less injured and exhibited greater root length and growth compared with the WT. It was concluded that the amount of injury to the wheat plants was negatively correlated with the level of accumulation of glycine betaine, and the glycine betaine acted as an important osmoprotectant in transgenic plants to improve root growth, and enhance the resistance of transgenic plants to drought stress.

A putative glutamate decarboxylase (GAD) gene, designated ZmGAD1, was cloned from Zea mays with a combination of reverse-transcriptase polymerase chain reaction (RT-PCR) and bioinformatic approaches. The ZmGAD1 cDNA sequence contained a complete open reading frame encoding a putative protein of 496 amino acids, which contained a pyridoxal-5′-phosphate binding domain and a calmodulin (CaM)-binding domain found in nearly all GADs from plants. Sequence analysis showed that it had highest similarity with rice GAD1. Recombinant ZmGAD1 protein was expressed in Escherichia coli, purified and used to measure enzyme activity, which confirmed ZmGAD1 was really a glutamate decarboxylase gene. Southern blotting analysis suggested that ZmGAD1 was present as a single copy gene in the maize genome. RT-PCR analysis revealed that ZmGAD1 was expressed in all examined tissues including the roots, stems, leaves, ears, and tassels. The expression of the ZmGAD1 gene was upregulated and GAD activity was increased in the leaves and roots after treatment with ABA, MeJA, NaCl, PEG, or cold stress. Several stress-related cis-elements were present in the ZmGAD1 promoter cloned from maize genomic DNA. These results suggested that ZmGAD1 might play an important role in responses to abiotic factors and hormone treatments.

The DREB/CBF transcription factors play important roles during low temperature, drought, and high-salt stress in higher plants. In this paper, we transferred TsCBF1 gene from a dicotyledonous halophyte Thellungiella halophila into the monocotyledonous crop maize (Zea mays L.). PCR, Southern blot, and RT-PCR analysis indicated that the TsCBF1 gene had been integrated into the genome of transgenic plants and was expressed in their progeny. After 14 days of drought stress treatments, transgenic plants showed improved drought tolerance with higher Relative Water Content (RWC), higher solute accumulation, and less cell damage compared with wild-type (WT) plants. Most importantly, they showed shorter anthesis-silking interval (ASI) and produced much higher grain yield than WT under drought stress. The results indicate that the TsCBF1 gene conferred enhanced drought tolerance to maize plants and may have utility for improving tolerance to other abiotic stresses as well.

Previous studies have shown that the overexpression of betA (encoding choline dehydrogenase from Escherichia coli) or AtNHX1 (a vacuolar Na(+)/H(+) antiport from Arabidopsis thaliana) gene can improve the salt tolerance of transgenic plants. However, little is known about the effects of the transgene pyramiding of betA and AtNHX1. Here, betA + AtNHX1 transgene pyramiding tobacco was produced by sexual crossing, and the salt tolerance was evaluated at the cellular and plant levels. In NaCl stress, the Na(+) concentration in vacuoles and vacuolar membrane potential of transgene pyramiding cells were similar to those of AtNHX1-transgenics, and much higher than those of betA-transgenics when detected using fluorescent dye staining; transgene pyramiding cells showed a higher protoplast viability and comparable mitochondrial activity as compared with single transgenics; and transgene pyramiding plants showed comparable Na(+) content in leaves as compared with AtNHX1-transgenics and remarkably higher than betA-transgenics; and transgene pyramiding lines exhibited higher percentage of seed germination, better seedling growth and higher fresh weight than lines that had betA or AtNHX1 alone. Based on the integrative analysis of salt tolerance, the consistency between the cellular level and the whole plant level was confirmed and the transgene pyramiding plants exhibited improved salt tolerance, but compared with the plants with betA or AtNHX1 alone, the differences were relatively small. Other mechanisms involved in salt tolerance should be considered to further enhance transgene pyramiding plants salt tolerance.

An H(+)-PPase gene, TsVP from Thellungiella halophila, was transferred into two cotton (Gossypium hirsutum) varieties (Lumianyan19 and Lumianyan 21) and southern and northern blotting analysis showed the foreign gene was integrated into the cotton genome and expressed. The measurement of isolated vacuolar membrane vesicles demonstrated that the transgenic plants had higher V-H(+)-PPase activity compared with wild-type plants (WT). Overexpressing TsVP in cotton improved shoot and root growth, and transgenic plants were much more resistant to osmotic/drought stress than the WT. Under drought stress conditions, transgenic plants had higher chlorophyll content, improved photosynthesis, higher relative water content of leaves and less cell membrane damage than WT. We ascribe these properties to improved root development and the lower solute potential resulting from higher solute content such as soluble sugars and free amino acids in the transgenic plants. In this study, the average seed cotton yields of transgenic plants from Lumianyan 19 and Lumianyan 21 were significantly increased compared with those of WT after exposing to drought stress for 21 days at flowering stage. The average seed cotton yields were 51 and 40% higher than in their WT counterparts, respectively. This study benefits efforts to improve cotton yields in arid and semiarid regions.

A novel flavin-containing monooxygenase (FMO)-like gene, designated as TsFMO, was cloned from Thellungiella halophila by reverse-transcriptase polymerase chain reaction (RT-PCR) coupled with rapid amplification of cDNA ends approach. The cDNA sequence of TsFMO had a complete open reading frame encoding a putative protein of 461 amino acids, which contained the conserved domains of FMOs. Sequence analysis showed that it had highest similarity with Arabidopsis FMOGS-OX1. RT-PCR analysis revealed that TsFMO was constitutively expressed in all examined tissues including the roots and rosette leaves of the seedlings and mature plants, bolts, and open flowers. Real-time PCR suggested that the gene was quickly and significantly upregulated in the leaves exposed to NaCl stress, which showed that TsFMO might be regulated with the redox state of plant cell. TsFMO was found to complement and rescue the retardant growth to dithiothreitol of yeast fmo mutant strain. In addition, TsFMO was expressed in Escherichia coli and the recombinant protein was purified to produce polyclonal antibody. Western blot with T. halophila extracts showed that the polyclonal antibody was effective and specific.

Low phosphorus (P) availability is a major limitation for plant growth. To better understand the molecular mechanism of P efficiency in maize, comparative proteome analyses were performed on the roots of the low-P-tolerant mutant 99038 and wild-type Qi-319 grown under P-sufficient (+P) or P-deficient (-P) conditions. Over 10% of proteins detected on two-dimensional electrophoresis (2-DE) gels showed expression that was altered twofold or more between the genotypes under +P or -P conditions. We identified 73 (+P) and 95 (-P) differentially expressed proteins in response to phosphate (Pi) starvation. These proteins were involved in a large number of cellular and metabolic processes, with an obvious functional skew toward carbon metabolism and regulation of cell proliferation. Further analysis of proteome data, physiological measurements and cell morphological observations showed that, compared to the wild-type, the low-P-tolerant mutant could accumulate and secrete more citrate under Pi starvation, which facilitates solubilization of soil Pi and enhances Pi absorption. The proportion of sucrose in the total soluble sugars of the low-P-tolerant mutant was significantly higher, and cell proliferation in root meristem was accelerated. This resulted in better developed roots and more advantageous root morphology for Pi uptake. These results indicate that differences in citrate secretion, sugar metabolism and root-cell proliferation are the main reasons for higher tolerance to low-P conditions in the mutant compared to the wild-type. Thus, the mutant displayed specialized P-efficient root systems with a higher capacity for mobilization of external Pi and increased cell division in the root meristem under Pi starvation.

Salinity is one of the major environmental factors limiting plant growth and productivity. An H(+)-PPase gene, TsVP from Thellungiella halophila, was transferred into cotton (Gossypium hirsutum) in sense and antisense orientations under control of the cauliflower mosaic virus (CaMV) 35S promoter. Southern and Northern blotting analysis showed that the sense or antisense TsVP were integrated into the cotton genome and expressed. Transgenic plants overexpressing the vacuolar H(+)-PPase were much more resistant to 150 and 250 mM NaCl than the isogenic wild-type plants. In contrast, the plants from the antisense line (L-2), with lower H(+)-PPase activity, were more sensitive to salinity than the wild-type plants. Overexpressing TsVP in cotton improved shoot and root growth and photosynthetic performance. These transgenic plants accumulated more Na(+), K(+), Ca(2+), Cl(-) and soluble sugars in their root and leaf tissues under salinity conditions compared with the wild-type plants. The lower membrane ion leakage and malondialdehyde (MDA) level in these transgenic plants suggest that overexpression of H(+)-PPase causes the accumulation of Na(+) and Cl(-) in vacuoles instead of in the cytoplasm, thus reducing their toxic effects. On the other hand, the increased accumulation of ions and sugars decreases the solute potential in cells, and facilitates water uptake under salinity, which is an important mechanism for the increased salt tolerance in TsVP-overexpressing cotton.

The vacuolar H(+)-translocating inorganic pyrophosphatase (H(+)-PPase) uses pyrophosphate as substrate to generate the proton electrochemical gradient across the vacuolar membrane to acidify vacuoles in plant cells. The heterologous expression of H(+)-PPase genes (TsVP from Thellungiella halophila and AVP1 from Arabidopsis thaliana) improved the salt tolerance of tobacco plants. Under salt stress, the transgenic seedlings showed much better growth and greater fresh weight than wild-type plants, and their protoplasts had a normal appearance and greater vigor. The cytoplasmic and vacuolar pH in transgenic and wild-type cells were measured with a pH-sensitive fluorescence indicator. The results showed that heterologous expression of H(+)-PPase produced an enhanced proton electrochemical gradient across the vacuolar membrane, which accelerated the sequestration of sodium ions into the vacuole. More Na(+) accumulated in the vacuoles of transgenic cells under salt (NaCl) stress, revealed by staining with the fluorescent indicator Sodium Green. It was concluded that the tonoplast-resident H(+)-PPase plays important roles in the maintenance of the proton gradient across the vacuolar membrane and the compartmentation of Na(+) within vacuoles, and heterologous expression of this protein enhanced the electrochemical gradient across the vacuolar membrane, thereby improving the salt tolerance of tobacco cells.

The tolerance to drought stress of the homozygous transgenic cotton (Gossypium hirsutum L.) plants with enhanced glycinebetaine (GB) accumulation was investigated at three development stages. Among the five transgenic lines investigated, lines 1, 3, 4, and 5 accumulated significantly higher levels of GB than the wild-type (WT) plants either before or after drought stress, and the transgenic plants were more tolerant to drought stress than the wild-type counterparts from young seedlings to flowering plants. Under drought stress conditions, transgenic lines 1, 3, 4, and 5 had higher relative water content, increased photosynthesis, better osmotic adjustment (OA), a lower percentage of ion leakage, and less lipid membrane peroxidation than WT plants. The GB levels in transgenic plants were positively correlated with drought tolerance under water stress. The results suggested that GB may not only protect the integrity of the cell membrane from drought stress damage, but also be involved in OA in transgenic cotton plants. Most importantly, the seedcotton yield of transgenic line 4 was significantly greater than that of WT plants after drought stress, which is of great value in cotton production.

The study of maize rough dwarf disease (MRDD) and breeding for resistance requires inoculation of maize plants by means of planthoppers. The plant age, insect density and inoculation duration are main factors in the success of maize rough dwarf disease inoculation. These parameters were tested using a susceptible maize inbred line Ye478. Using one or two-leaf plants, 15 planthoppers per plant and a five day inoculation duration, the line Ye478 was the most susceptible with 100% diseased plants; F112132 was moderately susceptible with 60% diseased plants and 90110 and F022411 were resistant without any disease. The results were consistent with those from six years of field studies. Using enzyme-linked immunosorbent assay (ELISA) and real-time quantitative RT-PCR, rice black-streaked dwarf virus was detected in severely diseased plants. The plants were rated from 0 to 3 according to their symptoms at the time of flowering. Plants scoring 0, 1 and 2 could not be distinguished by ELISA, only by real-time quantitative RT-PCR. All of the plants with a score of 3 were positive by ELISA and real-time quantitative RT-PCR. The significant differences in the average viral contents in plants with different symptom ratings could be distinguished by using real-time RT-PCR.

An H(+)-pyrophosphatase (PPase) gene named TsVP involved in basic biochemical and physiological mechanisms was cloned from Thellungiella halophila. The deduced translation product has similar characteristics to H(+)-PPases from other species, such as Arabidopsis and rice, in terms of bioinformation. The heterologous expression of TsVP in the yeast mutant ena1 suppressed Na(+) hypersensitivity and demonstrated the function of TsVP as an H(+)-PPase. Transgenic tobacco overexpressing TsVP had 60% greater dry weight than wild-type tobacco at 300 mM NaCl and higher viability of mesophyll protoplasts under salt shock stress conditions. TsVP and AVP1, another H(+)-PPase from Arabidopsis, were heterologously expressed separately in both the yeast mutant ena1 and tobacco. The salt tolerance of TsVP or AVP1 yeast transformants and transgenic tobacco were improved to almost the same level. The TsVP transgenic tobacco lines TL3 and TL5 with the highest H(+)-PPase hydrolytic activity were studied further. These transgenic tobacco plants accumulated 25% more solutes than wild-type plants without NaCl stress and 20-32% more Na(+) under salt stress conditions. Although transgenic tobacco lines TL3 and TL5 accumulated more Na(+) in leaf tissues, the malondialdehyde content and cell membrane damage were less than those of the wild type under salt stress conditions. Presumably, compartmentalization of Na(+) in vacuoles reduces its toxic effects on plant cells. This result supports the hypothesis that overexpression of H(+)-PPase causes the accumulation of Na(+) in vacuoles instead of in the cytoplasm and avoids the toxicity of excessive Na(+) in plant cells.

This study was carried out to evaluate the effects of purine synthesis inhibitor mizoribine, purine and pyrimidine synthesis inhibitors azaserine and acivicin, and surfactant Silwet L-77 on Agrobacterium-mediated transformation efficiency of embryogenic calluses from maize elite inbred lines Qi 319 and Ye 515. After transformation and three rounds of selection on 2.8 μM chlorsulfuron, resistant calluses were obtained subsequently, and morphologically normal plantlets were regenerated from 80 to 90% of the resistant calluses treated with the compounds. There were no obvious discrepancies between the frequencies of plantlet regeneration and the ratio of PCR positive plantlets of calluses treated with different compounds. Results of PCR assay with primers for betA showed that 40.2% (103/256) of the regenerated plantlets were positive. The percentage of resistant calluses was 2–3-fold higher than the control after being treated with 0.19–0.27 mM mizoribine. The most suitable concentration of azaserin was 0.36 mM, which gave a 4-fold increase in the percentage of resistant calluses. Acivicin at 0.28–0.84 mM yielded a 3–5-fold increase in the percentage of resistant calluses, which is significantly better than the control. When the calluses were treated with 0.01 or 0.02% Silwet L-77, the percentages of resistant calluses were 34.89 and 25.60%, respectively. We concluded that purine synthesis inhibitors, purine and pyrimidine synthesis inhibitor and the surfactant Silwet L-77 at optimal concentrations significantly improved the Agrobacterium-mediated transformation of maize calluses.

The inheritance and stability of the acetolactate synthase (als) transgene were compared in transgenic maize plants, generated using the pollen-tube pathway, particle bombardment, or Agrobacterium-mediated methods of transformation. Progeny populations generated by successive selfing or backcrossing of primary transformants were analyzed over three generations, using PCR and herbicide screening, to examine segregation and als activity, respectively, and transgenic homozygous plants were selected. The pollen-tube method resulted in a higher rate of primary normal transgenic plants and a less-stable transmission of the als locus than did the other two methods. When transferred by the particle bombardment and Agrobacterium-mediated methods, the als gene was in a much higher proportion of Mendelian transmission than transferred by the pollen-tube method. Compared to the Agrobacterium-mediated transformation, the particle bombardment method tends to create multiple copies and insert sites of the als gene in maize genome, which delaying the homogenization of the als locus with advancing generations. Agrobacterium-mediated transformation resulted in a greater proportion of stable, low copy number (in general 1–2) transgenic events, facilitating the stable inheritance of the als gene, and producing multiple desirable transgenic plants.

Glycine betaine plays an important role in some plants, including maize, in conditions of abiotic stress, but different maize varieties vary in their capacity to accumulate glycine betaine. An elite maize inbred line DH4866 was transformed with the betA gene from Escherichia coli encoding choline dehydrogenase (EC 1.1.99.1), a key enzyme in the biosynthesis of glycine betaine from choline. The transgenic maize plants accumulated higher levels of glycine betaine and were more tolerant to drought stress than wild-type plants (non-transgenic) at germination and the young seedling stage. Most importantly, the grain yield of transgenic plants was significantly higher than that of wild-type plants after drought treatment. The enhanced glycine betaine accumulation in transgenic maize provides greater protection of the integrity of the cell membrane and greater activity of enzymes compared with wild-type plants in conditions of drought stress.

The betA gene from Escherichia coli encoding choline dehydrogenase was transferred into elite maize inbred DH4866 via Agrobacterium -mediated transformation. Among five transgenic maize lines investigated, four lines, lines 2, 3, 4 and 5, accumulated significantly higher levels of glycinebetaine than wild type plants (non-transformants). At 10 and 15 °C, transgenic lines 2, 3, 4 and 5 exhibited higher germination capacity as determined by the germination speed and final germination percentage, and displayed less inhibition in seedling shoot growth rate than wild type plants. In chilling conditions, the chilling tolerance, expressed in cell membrane damage, degree of chilling injury, survival rate, and photosynthesis was enhanced in plants of transgenic lines 2, 3, 4 and 5 as compared with wild type plants. The increase of total soluble sugars and the increase of total free amino acids that was mostly due to accumulation of alanine, glutamic acid, glycine and serine might play an important role of osmoregulation under stress conditions.

Maize inbred line 77Ht2 contains agriculturally important genes and has been widely used in corn breeding in China. A bacterial artificial chromosome (BAC) library of 77Ht2 has been constructed in order to identify useful genes and to facilitate the study of the maize genome. The library contains 175104 clones with an average insert size of 57 kb and represents about 4 maize haploid genome equivalents. Characterization of the library showed less than 0.5% of clones to not contain large inserts. Significant contamination of chloroplast and mitochondria DNA was not detected. BAC clones (152 arrays) were stored in 96 microtiter plates, with each well containing 12 clones. This is the first maize BAC library constructed in China. It is well suited for map-based cloning of maize genes and genome physical mapping.

Phosphatidylinositol (PtdIns) is an important lipid because it serves as a key membrane constituent and is the precursor of the inositol-containing lipids that are found in all plants and animals. It is synthesized from cytidine-diphosphodiacylglycerol (CDP-DG) and myo-inositol by PtdIns synthase (PIS). We have previously reported that two putative PIS genes from maize (Zea mays L.), ZmPIS and ZmPIS2, are transcriptionally up-regulated in response to drought (Sui et al., Gene, 426:47-56, 2008). In this work, we report on the characterization of ZmPIS in vitro and in vivo. The ZmPIS gene successfully complemented the yeast pis mutant BY4743, and the determination of PIS activity in the yeast strain further confirmed the enzymatic function of ZmPIS. An ESI-MS/MS-based lipid profiling approach was used to identify and quantify the lipid species in transgenic and wild-type tobacco plants before and after drought treatment. The results show that the overexpression of ZmPIS significantly increases lipid levels in tobacco leaves under drought stress compared to those of wild-type tobacco, which correlated well with the increased drought tolerance of the transgenic plants. Further analysis showed that, under drought stress conditions, ZmPIS overexpressors were found to exhibit increased membrane integrity, thereby enabling the retention of more solutes and water compared with the wild-type and the vector control transgenic lines. Our findings give us new insights into the role of the ZmPIS gene in the response of maize to drought/osmotic stress and the mechanisms by which plants adapt to drought stress.

A bHLH (basic helix-loop-helix domain) transcription factor involved in tolerance to Pi starvation was cloned from Zea mays with an RT-PCR coupled RACE approach and named ZmPTF1. ZmPTF1 encoded a putative protein of 481 amino acids that had identity with OsPTF1 in basic region. Real-time RT-PCR revealed that ZmPTF1 was quickly and significantly up-regulated in the root under phosphate starvation conditions. Overexpression of ZmPTF1 in maize improved root development, enhanced biomass both in hydroponic cultures and sand pots, and the plants developed more tassel branches and larger kernels when they were grown in low phosphate soil. Compared with wild type, overexpressing ZmPTF1 altered the concentrations of soluble sugars in transgenic plants, in which soluble sugars levels were lower in the leaves and higher in the roots. Overexpression of ZmPTF1 enhanced the expression of fructose-1,6-bisphosphatase and sucrose phosphate synthase1 participated in sucrose synthesis in the leaves but decreased them in the root, and reduced the expression of genes involved in sucrose catabolism in the roots. The modifications on the physiology and root morphology of the plants enhanced low phosphate tolerance and increased the yield under low phosphate conditions. This research provides a useful gene for transgenic breeding of maize that is tolerant to phosphate deficiency and is helpful for exploring the relationship between sugar signaling and phosphate concentrations in cells.

Cereal crops accumulate starch in the seed endosperm as an energy reserve. ADP-glucose pyrophosphorylase (AGPase) plays a key role in regulating starch biosynthesis in cereal seeds. The AGPase in the maize endosperm is a heterotetramer of two small subunits, encoded by Brittle2 (Bt2) gene, and two large subunits, encoded by the Shrunken2 (Sh2) gene. The two genes (Bt2, Sh2) from maize were introduced into two elite maize inbred lines, solely and in tandem, and under the control of endosperm-specific promoters for over-expression. PCR, Southern blotting, and real-time RT-PCR analysis indicated that the transgenes were integrated into the genome of transgenic plants and were over-expressed in their progeny. The over-expression of either gene enhanced AGPase activity, seed weight and starch content compared with the WT, but the amounts were lower than plants with over-expression of both Bt2 and Sh2. Developing seeds from co-expression transgenic maize plants had higher cytoplasmic AGPase activity: the 100-grain weight increased 15% over the wild type (WT), and the starch content increased to over 74% compared with the WT of 65%. These results indicate that over-expression of the genes in transgenic maize plants could improve kernel traits. This report provides a feasible approach for increasing starch content and seed weight in maize.

Background Salt stress is one of the major abiotic stresses affecting plant growth and productivity. Vacuolar H+-pyrophosphatase (H+-PPase) genes play an important role in salt stress tolerance in multiple species. Results In this study, the promoter from the vacuolar H+-pyrophosphatase from Thellungiella halophila (TsVP1) was cloned and compared with the AVP1 promoter from Arabidopsis thaliana. Sequence analysis indicated that these two promoters had seven similar motifs at similar positions. To determine which tissues the two promoters are active in, transgenic plants were produced with expression of the GUS reporter gene under the control of one of the promoters. In transgenic Arabidopsis with the TsVP1 promoter, the GUS reporter gene had strong activity in almost all tissues except the seeds and the activity was induced in both shoots and roots, especially in the root tips, when treated with salt stress. Such induction was not found in transgenic Arabidopsis with the AVP1 promoter. By analyzing different 5' deletion mutants of the TsVP1 promoter, an 856 bp region (-2200 to -1344) was found to contain enhancer elements that increased gene expression levels. Two AAATGA motifs, which may be the key elements for the anther specific expression profile, in the deleted TsVP1 promoters (PT2 to PT6) were also identified. A 130 bp region (-667 to -538) was finally identified as the key sequence for the salt stress response by analyzing the different mutants both with and without salt stress. GUS transient assay in tobacco leaves suggested the 130 bp region was sufficient for the salt stress response. Bioinformatic analysis also revealed that there may be novel motifs in this region that are the key elements for the salt stress responsive activity of the TsVP1 promoter. Conclusions The TsVP1 promoter had strong activity in almost all tissues except the seeds. In addition, its activity was induced by salt stress in leaves and roots, especially in root tips. A 130 bp region (-667 to -538) was identified as the key region for responding to salt stress.

Phytohormones play critical roles in regulating plant responses to stress. We investigated the effects of water stress induced by adding 12% (w/v) polyethylene glycol to the root medium on the levels of abscisic acid (ABA), indole-3-acid (IAA), zeatin (ZT), and gibberellin(3) (GA(3)) in maize leaves. The results suggested that water stress had significant effects on the four hormone levels. There was a transient increase in the IAA content during the initial stage of adaptation to water stress in maize leaves, but it dropped sharply thereafter in response to water stress. ABA content increased dramatically in maize leaves after 24 h of exposure to water stress, and then the high levels of ABA were maintained to the end. The contents of ZT and GA(3) rapidly declined in maize leaves subjected to water stress. The effects of water stress on chlorophyll content, electrolyte leakage and malondialdehyde levels in maize leaves were also studied. The variation of cell damage was negatively correlated with ZT and GA(3) levels in maize leaves under water stress. Thus, we explored the roles of ZT and GA(3) on the growth of maize seedlings under water stress by exogenous application. It is possible that both ZT and GA(3) were effective in protecting maize seedlings from water stress, which would be of great importance for the improvement of drought tolerance in maize by genetic manipulation.

In this study, it was shown that the TsVP gene [vacuolar H+-pyrophosphatase (V-H+-PPase) gene from a dicotyledonous halophyte Thellungiella halophila] could be transferred into the monocotyledonous crop maize (Zea mays L.), and that the heterologous expression of the transgene improved the drought resistance of transgenic plants. Polymerase chain reaction amplification and Southern blotting confirmed the existence of the foreign gene in transformed plants and their progeny. Expression differences of the TsVP gene in different transgenic lines were monitored by reverse transcriptase-polymerase chain reaction. The measurement of isolated vacuolar membrane vesicles from the TsVP transgenic and wild-type (WT) plants demonstrated that the transgenic plants had higher V-H+-PPase activity, and the performance of maize-expressed TsVP in response to osmotic/drought stress was better in lines with higher V-H+-PPase activity. Transgenic plants showed a higher percentage of seed germination, better developed root systems and greater biomass, greater solute accumulation and less cell membrane damage relative to WT plants under osmotic stress. After drought stress treatment, transgenic plants showed less growth retardation and shorter anthesis-silking interval, and produced much larger grain yields, than WT plants. It was concluded that the high V-H+-PPase activity of transgenic maize improved the drought resistance of plants. This report provides a feasible approach to increase monocotyledonous crop yields under conditions of soil water deficit by the heterologous expression of TsVP.

Phosphatidylinositol-specific phospholipase C (PI-PLC) plays an important role in a variety of physiological processes in plants, including drought tolerance. It has been reported that the ZmPLC1 gene cloned from maize (Zea mays L.) encoded a PI-PLC and up-regulated the expression in maize roots under dehydration conditions (Zhai SM, Sui ZH, Yang AF, Zhang JR in Biotechnol Lett 27:799-804, 2005). In this paper, transgenic maize expressing ZmPLC1 transgenes in sense or antisense orientation were generated by Agrobacterium-mediated transformation and confirmed by Southern blot analysis. High-level expression of the transgene was confirmed by real-time RT-PCR and PI-PLC activity assay. The tolerance to drought stress (DS) of the homogenous transgenic maize plants was investigated at two developmental stages. The results demonstrated that, under DS conditions, the sense transgenic plants had higher relative water content, better osmotic adjustment, increased photosynthesis rates, lower percentage of ion leakage and less lipid membrane peroxidation, higher grain yield than the WT; whereas those expressing the antisense transgene exhibited inferior characters compared with the WT. It was concluded that enhanced expression of sense ZmPLC1 improved the drought tolerance of maize.

Water-deficit stress during meiosis is one of the most serious threats to crop production. To elucidate the mechanisms of the response to water-deficit stress in the reproductive organs of maize, we have characterized the changes in transcription that occur during meiosis in the tassels and floret formation in the ears following water deficit stress. We used oligo microarray analysis, which included 57,452 transcripts representing more than 30,000 identifiable unique maize genes, and combined this with reverse Northern blot analysis. After 7 days of stress, immature tassels and ears differed considerably in their transcriptional responses, and the majority of changes were organ specific. In the tassels, 1,513 transcripts were differentially expressed (by threefold or greater) with 62% of these being upregulated by water stress. In the ears, 202 transcripts were differentially expressed with 95% being upregulated by water stress. Most of these transcripts have not been previously reported to be associated with water stress. Only 74 of these transcripts were co-regulated in the two organs. The stress-regulated transcripts are involved in a broad range of cellular and biochemical activities. The most notable may function in carbohydrate metabolism, particular in sucrose, trehalose and raffinose metabolism, and in cell wall metabolism in the tassels. Collectively, these data suggest that the transcripts differentially expressed during reproductive organic development may represent candidate genes for dissecting molecular mechanism of this important biological process in response to water-deficit stress.

Improved drought tolerance is always a highly desired trait for agricultural plants. Significantly increased drought tolerance in Arabidopsis thaliana (Columbia-0) has been achieved in our work through the suppression of ESKMO1 (ESK1) gene expression with small-interfering RNA (siRNA) and overexpression of CBF genes with constitutive gene expression. ESK1 has been identified as a gene linked to normal development of the plant vascular system, which is assumed directly related to plant drought response. By using siRNA that specifically targets ESK1, the gene expression has been reduced and drought tolerance of the plant has been enhanced dramatically in the work. However, the plant response to external abscisic acid application has not been changed. ICE1, CBF1, and CBF3 are genes involved in a well-characterized plant stress response pathway, overexpression of them in the plant has demonstrated capable to increase drought tolerance. By overexpression of these genes combining together with suppression of ESK1 gene, the significant increase of plant drought tolerance has been achieved in comparison to single gene manipulation, although the effect is not in an additive way. Accompanying the increase of drought tolerance via suppression of ESK1 gene expression, the negative effect has been observed in seeds yield of transgenic plants in normal watering conditions comparing with wide type plant.

Background Phosphorus (P) is an essential macronutrient for all living organisms. Maize (Zea mays) is an important human food, animal feed and energy crop throughout the world, and enormous quantities of phosphate fertilizer are required for maize cultivation. Thus, it is important to improve the efficiency of the use of phosphate fertilizer for maize. Results In this study, we analyzed the maize root response to phosphate starvation and performed a transcriptomic analysis of the 1.0-1.5 cm lateral root primordium zone. In the growth of plants, the root-to-shoot ratio (R/L) was reduced in both low-phosphate (LP) and sufficient-phosphate (SP) solutions, but the ratio (R/L) exhibited by the plants in the LP solution was higher than that of the SP plants. The growth of primary roots was slightly promoted after 6 days of phosphate starvation, whereas the numbers of lateral roots and lateral root primordia were significantly reduced, and these differences were increased when associated with the stress caused by phosphate starvation. Among the results of a transcriptomic analysis of the maize lateral root primordium zone, there were two highlights: 1) auxin signaling participated in the response and the modification of root morphology under low-phosphate conditions, which may occur via local concentration changes due to the biosynthesis and transport of auxin, and LOB domain proteins may be an intermediary between auxin signaling and root morphology; and 2) the observed retardation of lateral root development was the result of co-regulation of DNA replication, transcription, protein synthesis and degradation and cell growth. Conclusions These results indicated that maize roots show a different growth pattern than Arabidopsis under low-phosphate conditions, as the latter species has been observed to halt primary root growth when the root tip comes into contact with low-phosphate media. Moreover, our findings enrich our understanding of plant responses to phosphate deficits and of root morphogenesis in maize.