Proteomic analysis of shoot-borne root initiation in maize (Zea mays L.)

Postembryonically formed shoot-borne roots make up the major backbone of the adult maize root stock. In this study the abundant soluble proteins of the first node (coleoptilar node) of wild-type and mutant rtcs seedlings, which do not initiate crown roots, were compared at two early stages of crown root formation. In Coomassie Bluestained 2-D gels, representing soluble proteins of coleoptilar nodes 5 and 10 days after germination, 146 and 203 proteins were detected, respectively. Five differentially accumulated proteins (> two-fold change; t-test: 95% significance) were identified in 5-day-old and 14 differentially accumulated proteins in 10-day-old coleoptilar nodes of wild-type versus rtcs. All 19 differentially accumulated proteins were identified via ESI MS/MS mass spectrometry. Five differentially accumulated proteins, including a regulatory G-protein and a putative auxin-binding protein, were further analyzed at the RNA expression level. These experiments confirmed differential gene expression and revealed subtle developmental regulation of these genes during early coleoptilar node development. This study represents the first proteomic analysis of shoot-borne root initiation in cereals and will contribute to a better understanding of the molecular basis of this developmental process unique to cereals.     

Rootless with undetectable meristem 1 encodes a monocot-specific AUX/IAA protein that controls embryonic seminal and post-embryonic lateral root initiation in maize

The maize (Zea mays L.) rum1‐R (rootless with undetectable meristems 1‐Reference) mutant does not initiate embryonic seminal roots and post‐embryonic lateral roots at the primary root. Map‐based cloning revealed that Rum1 encodes a 269 amino acid (aa) monocot‐specific Aux/IAA protein. The rum1‐R protein lacks 26 amino acids including the GWPPV degron sequence in domain II and part of the bipartite NLS (nuclear localization sequence). Significantly reduced lateral root density (approximately 35%) in heterozygous plants suggests that the rum1‐R is a semi‐dominant mutant. Overexpression of rum1‐R under the control of the maize MSY (Methionine SYnthase) promoter supports this notion by displaying a reduced number of lateral roots (31–37%). Functional characterization suggests that Rum1 is auxin‐inducible and encodes a protein that localizes to the nucleus. Moreover, RUM1 is unstable with a half life time of approximately 22 min while the mutant rum1‐R protein is very stable. In vitro and in vivo experiments demonstrated an interaction of RUM1 with ZmARF25 and ZmARF34 (Z. mays AUXIN RESPONSE FACTOR 25 and 34). In summary, the presented data suggest that Rum1 encodes a canonical Aux/IAA protein that is required for the initiation of embryonic seminal and post‐embryonic lateral root initiation in primary roots of maize.     

Molecular interactions of ROOTLESS CONCERNING CROWN AND SEMINAL ROOTS, a LOB domain protein regulating shoot-borne root initiation in maize (Zea mays L.)

Rootless concerning crown and seminal roots (Rtcs) encodes a LATERAL ORGAN BOUNDARIES domain (LBD) protein that regulates shoot-borne root initiation in maize (Zea mays L.). GREEN FLUORESCENT PROTEIN (GFP)-fusions revealed RTCS localization in the nucleus while its paralogue RTCS-LIKE (RTCL) was detected in the nucleus and cytoplasm probably owing to an amino acid exchange in a nuclear localization signal. Moreover, enzyme-linked immunosorbent assay (ELISA) experiments demonstrated that RTCS primarily binds to LBD DNA motifs. RTCS binding to an LBD motif in the promoter of the auxin response factor (ARF) ZmArf34 and reciprocally, reciprocal ZmARF34 binding to an auxin responsive element motif in the promoter of Rtcs was shown by electrophoretic mobility shift assay experiments. In addition, comparative qRT-PCR of wild-type versus rtcs coleoptilar nodes suggested RTCS-dependent activation of ZmArf34 expression. Consistently, luciferase reporter assays illustrated the capacity of RTCS, RTCL and ZmARF34 to activate downstream gene expression. Finally, RTCL homo- and RTCS/RTCL hetero-interaction were demonstrated in yeast-two-hybrid and bimolecular fluorescence complementation experiments, suggesting a role of these complexes in downstream gene regulation. In summary, the data provide novel insights into the molecular interactions resulting in crown root initiation in maize.     

Regulation of the maize (Zea mays L.) embryo proteome by RTCS which controls seminal root initiation

Seminal roots are initiated at the scutellar node during maize (Zea mays L.) embryo development. The maize mutant rtcs (rootless concerning crown and seminal roots) does not initiate seminal roots while its wild-type siblings form on average 2.9 seminal roots per seedling. In this study, proteome profiles of 25-day-old immature maize embryos were compared between wild-type and rtcs plants via two-dimensional electrophoresis (2-DE). Electrospray ionization tandem mass spectrometry (ESI-MS/MS) identified 23 proteins encoded by 21 different genes that were differentially accumulated between wild-type and rtcs embryos (Fc≥2; FDR<10%). Among the differentially accumulated proteins, two isoforms of a phosphoglycerate kinase and a malate dehydrogenase were preferentially accumulated in wild-type embryos. Both enzymes are related to the generation of energy-rich ATP or NADPH molecules and are crucial checkpoints of cellular energetics in plants. Comparison of embryonic proteins differentially accumulated between wild-type and rtcs embryos revealed little overlap with proteins differentially accumulated between wild-type and rum1 embryos which also do not initiate seminal roots. This might be due to distinct influences of RTCS and RUM1 on the composition of the embryo proteome, but could also be explained by different stages of embryo development that were analyzed in these studies.     

The maize (Zea mays L.) roothairless 3 gene encodes a putative GPI-anchored, monocot-specific, COBRA-like protein that significantly affects grain yield

The rth3 ( roothairless 3 ) mutant is specifically affected in root hair elongation. We report here the cloning of the rth3 gene via a PCR-based strategy (amplification of insertion mutagenized sites) and demonstrate that it encodes a COBRA-like protein that displays all the structural features of a glycosylphosphatidylinositol anchor. Genes of the COBRA family are involved in various types of cell expansion and cell wall biosynthesis. The rth3 gene belongs to a monocot-specific clade of the COBRA gene family comprising two maize and two rice genes. While the rice ( Oryza sativa ) gene OsBC1L1 appears to be orthologous to rth3 based on sequence similarity (86% identity at the protein level) and maize/rice synteny, the maize ( Zea mays L.) rth3-like gene does not appear to be a functional homolog of rth3 based on their distinct expression profiles. Massively parallel signature sequencing analysis detected rth3 expression in all analyzed tissues, but at relatively low levels, with the most abundant expression in primary roots where the root hair phenotype is manifested. In situ hybridization experiments confine rth3 expression to root hair-forming epidermal cells and lateral root primordia. Remarkably, in replicated field trials involving near-isogenic lines, the rth3 mutant conferred significant losses in grain yield.     

Genomic prediction of seedling root length in maize (Zea mays L.)

Genotypes with extreme phenotypes are valuable for studying ‘difficult’ quantitative traits. Genomic prediction (GP) might allow the identification of such extremes by phenotyping a training population of limited size and predicting genotypes with extreme phenotypes in large sequences of germplasm collections. We tested this approach employing seedling root traits in maize and the extensively genotyped Ames Panel. A training population made up of 384 inbred lines from the Ames Panel was phenotyped by extracting root traits from images using the software program aria . A ridge regression best linear unbiased prediction strategy was used to train a GP model. Genomic estimated breeding values for the trait ‘total root length’ (TRL) were predicted for 2431 inbred lines, which had previously been genotyped by sequencing. Selections were made for 100 extreme TRL lines and those with the predicted longest or shortest TRL were validated for TRL and other root traits. The two predicted extreme groups with regard to TRL were significantly different (P = 0.0001). The difference in predicted means for TRL between groups was 145.1 cm and 118.7 cm for observed means, which were significantly different (P = 0.001). The accuracy of predicting the rank between 1 and 200 of the validation population based on TRL (longest to shortest) was determined using a Spearman correlation to be ρ = 0.55. Taken together, our results support the idea that GP may be a useful approach for identifying the most informative genotypes in sequenced germplasm collections to facilitate experiments for quantitative inherited traits.     

Evaluation of parameters describing the root system architecture of field grown maize plants (Zea mays L.)

The objective of this work was to study elongation curves of maize axile roots throughout their elongation period under field conditions. Relationships between their elongation rate and the extension rate of their branched region were also studied. Maize, early-maturing cultivar Dea, was grown on a deep, barrier-free clay loam (depth 1.80m). Trenches were dug during four periods until after silking and axile roots were excavated. Parameters measured were total length and the lengths of basal and apical unbranched zones. The rank of the bearing phytomer and general data about the carrying plant were also recorded. Results showed that axile roots from lower phytomers had similar elongation rates irrespective of the rank of the carrying phytomer. This elongation rate declined with root age. A monomolecular elongation model was fitted to the experimental data. Elongation was much slower in roots from upper phytomers. A rough linear relationship was found between the elongation rate of axile roots and the length of the apical unbranched zone. This result suggests that laterals appeared on a root segment a constant time after it was formed. Possible mechanisms with may account for the declining elongation rate with root age (increasing distance from aerial parts or adverse environmental conditions in deep soil layers) and variability between individual roots are also discussed.     

Roothairless5, which functions in maize (Zea mays L.) root hair initiation and elongation encodes a monocot‐specific NADPH oxidase

Root hairs are instrumental for nutrient uptake in monocot cereals. The maize (Zea mays L.) roothairless5 (rth5) mutant displays defects in root hair initiation and elongation manifested by a reduced density and length of root hairs. Map‐based cloning revealed that the rth5 gene encodes a monocot‐specific NADPH oxidase. RNA‐Seq, in situ hybridization and qRT‐PCR experiments demonstrated that the rth5 gene displays preferential expression in root hairs but also accumulates to low levels in other tissues. Immunolocalization detected RTH5 proteins in the epidermis of the elongation and differentiation zone of primary roots. Because superoxide and hydrogen peroxide levels are reduced in the tips of growing rth5 mutant root hairs as compared with wild‐type, and Reactive oxygen species (ROS) is known to be involved in tip growth, we hypothesize that the RTH5 protein is responsible for establishing the high levels of ROS in the tips of growing root hairs required for elongation. Consistent with this hypothesis, a comparative RNA‐Seq analysis of 6‐day‐old rth5 versus wild‐type primary roots revealed significant over‐representation of only two gene ontology (GO) classes related to the biological functions (i.e. oxidation/reduction and carbohydrate metabolism) among 893 differentially expressed genes (FDR <5%). Within these two classes the subgroups ‘response to oxidative stress’ and ‘cellulose biosynthesis’ were most prominently represented.     

玉米幼苗根系分泌物对芘污染的响应

根际袋土培试验研究了玉米幼苗根系分泌物中的可溶性糖、低分子量有机酸和氨基酸对不同芘污染水平(50、200、800mgkg-1,记为T1、T2、T3)的响应差异,探讨芘胁迫下植物根系的生理生态效应。结果表明,较低浓度芘可适当地刺激玉米的生长,高浓度芘处理抑制了玉米的生长,并且抑制作用随芘处理浓度的提高而增强;芘对玉米根系的影响要大于对茎叶的影响。芘胁迫下促进了根系分泌可溶性糖、低分子量有机酸和氨基酸增多。T1、T2和T3处理根系分泌物中可溶性糖、低分子量有机酸、氨基酸含量分别是T0处理的1.14、1.81、1.35倍,1.24、4.31、2.94倍,1.58、5.56、5.40倍。不同芘污染水平下,乙酸分泌量表现为T2T3T1,酒石酸和柠檬酸分泌量表现为T3T2T1,草酸分泌量表现为T3≈T2T1。芘处理对根系分泌氨基酸种类的影响不大,而对各氨基酸分泌量的变化幅度影响较大;芘胁迫处理对于18种常见氨基酸组分的分泌量的影响各不相同。不同芘污染水平下,天门冬氨酸、丝氨酸和丙氨酸分泌量表现为T3T2T1,苏氨酸、谷氨酸、脯氨酸、甘氨酸、胱氨酸、缬氨酸、甲硫氨酸、异亮氨酸、亮氨酸、酪氨酸、苯丙氨酸、赖氨酸、组氨酸、γ-氨基丁酸、鸟氨酸分泌量表现为T2T3T1。     

Effect of environment on the early steps of ear initiation in maize (Zea mays L.)

The effects of environmental conditions on ear-shoot initiation have been investigated in three inbred genotypes of Zea mays L. which are used for seed production. Scanning electron microscopy (SEM) and binocular examination during the vegetative phase showed that axillary meristems are initiated at the same rate as the leaf primordia on the apical meristem, but with a delay of 5.6–7.0 plastochrons, depending on the genotype. Furthermore, the topmost axillary meristem is initiated on the same day as the tassel, whatever the genotype. One of the inbreds (B22) used in this study has been reported to exhibit, in field conditions, a reproductive failure affecting car initiation, causing the topmost car to be replaced at maturity by a sterile, leaf-like, structure. Scanning electron microscopic study of the formation of the abnormal axillary buds indicated that ear failure resulted from the early collapse of the axillary meristem followed by elongation of the prophyll or of the meristem itself. Using controlled environments, ear abortion was mimicked by a chilling treatment (10°C), given just before tassel initiation. Other factors, such as high irradiance and flooding, enhanced the abortive response. The critical stage for the main car was just before the initiation of the topmost axillary meristem which also corresponded to tassel initiation. Chilling the plants before or after tassel initiation either induced an acclimation response or had no effect. The three inbreds showed differential responses to the stress treatment, indicating that a genetic factor is implicated as well. It is suggested that chilling causes a perturbation of apical dominance which, in the responsive genotypes, represses axillary meristem development. The use of a stress-sensitive inbred such as B22 as a model system could yield some interesting clues to the mechanism of endogenous control of ear initiation in maize.     

玉米农田土壤呼吸作用的空间异质性及其根系呼吸作用的贡献

基于4月底到9月底东北地区玉米农田土壤呼吸作用全生长季的观测,阐明了土壤呼吸作用的空间异质性特征,综合分析了水热因子、土壤性质、根系生物量及其测定位置对土壤呼吸作用空间异质性的影响,并对生长季中根系呼吸作用占土壤呼吸作用的比例进行了估算。结果表明,在植株尺度上,土壤呼吸作用存在着明显的空间异质性,较高的土壤呼吸速率通常出现在靠近玉米植株的地方。根系生物量的分布格局是影响土壤呼吸作用空间异质性的关键因素。在空间尺度上,土壤呼吸作用与根系生物量呈显著的线性关系,而土壤湿度、土壤有机质、全氮和碳氮比对土壤呼吸作用空间异质性的影响并不显著。通过建立土壤呼吸作用与玉米根系生物量的回归方程,对根系呼吸作用占土壤呼吸作用的比例进行了间接估算。玉米生长季中,根系呼吸作用占土壤呼吸作用的比例在43．1％～63．6％之间波动,均值为54．5％。     

The effect of soil aggregate size on early shoot and root growth of maize (Zea mays L.)

Differences in plant growth arising from differences in aggregate size in the seedbed are normally atributed to limitations in nutrient or water supply during the early growth period. This study was initiated to determine if these were the only mechanisms by which aggregate size influences plant response. Four different aggregate size fractions (less than 1.6 mm, 1.6 to 3.2 mm, 3.2 to 6.4 mm and 6.4 to 12.8 mm diameter) were sieved from a silt loam soil. Nutrients were added to the soil and maize was grown in the aggregates for eighteen days after seedling emergence. Soil matric potential was maintained between — 3 and −20 kPa. Shoot dry weight declined by 18% as aggregate size increased from less than 1.6 mm to 1.6–3.2 mm. There was little further decline as aggregate size increased to 6.4–12.8 mm. Final leaf area showed a similar decline. The availability of nutrients or water were not limiting. Total root length in the coarsest aggregate system was less than 60% of that in the finest system. Main axes of seminal and nodal roots were longer in the coarser aggregate systems, the length of primary laterals was not affected, and length of secondary laterals was lower in the coarser systems. A greater proportion of the roots penetrated the larger aggregates than the smaller aggregates; however, the larger aggregates offered greater resistance to penetration by a rigid micropenetrometer (150 μ diameter probe). Diameter of the main axes roots were greatest in the largest two aggregate fractions. it is speculated that a combination of increased endogenous ethylene in roots in the finest aggregate system due to entrapment by water and increased mechanical resistance in the coarsest aggregate system accounts for the observed effects on root norphology.     

Comparative proteome analyses of phosphorus responses in maize (Zea mays L.) roots of wild-type and a low-P-tolerant mutant reveal root characteristics associated with phosphorus efficiency

SUMMARY: 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.     

Chlorophyll-deficient gene and morphological variations in Korean populations of maize (Zea mays)

We studied cultivated and naturalized Korean maize populations to determine the extent to which the chlorophylldeficient mutation and the phenotypic variations of two morphological characters (i.e., red coleoptiles and epicotyls, and the number of the first root hairs) are maintained. The frequency of the chlorophyll-deficient mutant gene (2.73% on average) was highly variable. Frequencies of red coleoptiles and epicotyls also were higher than expected from a mutation-selection balance. The average number of hairy phenotypes within populations was 1.8, ranging from 0.0 to 4.0. Naturalized populations were closely related to with cultivated communities. Most striking, however, was the more significant difference among populations than within populations with regard to both the frequency of chlorophyll-deficient mutant genes and the phenotypic variations of our two morphological characters. On a per-gene basis, the majority of the phenotypic variation (mean of 73.3%) resided among populations.     

A key to totipotency: Wuschel-like homeobox 2a unlocks embryogenic culture response in maize (Zea mays L.)

The ability of plant somatic cells to dedifferentiate, form somatic embryos and regenerate whole plants in vitro has been harnessed for both clonal propagation and as a key component of plant genetic engineering systems. Embryogenic culture response is significantly limited, however, by plant genotype in most species. This impedes advancements in both plant transformation-based functional genomics research and crop improvement efforts. We utilized natural variation among maize inbred lines to genetically map somatic embryo generation potential in tissue culture and identify candidate genes underlying totipotency. Using a series of maize lines derived from crosses involving the culturable parent A188 and the non-responsive parent B73, we identified a region on chromosome 3 associated with embryogenic culture response and focused on three candidate genes within the region based on genetic position and expression pattern. Two candidate genes showed no effect when ectopically expressed in B73, but the gene Wox2a was found to induce somatic embryogenesis and embryogenic callus proliferation. Transgenic B73 cells with strong constitutive expression of the B73 and A188 coding sequences of Wox2a were found to produce somatic embryos at similar frequencies, demonstrating that sufficient expression of either allele could rescue the embryogenic culture phenotype. Transgenic B73 plants were regenerated from the somatic embryos without chemical selection and no pleiotropic effects were observed in the Wox2a overexpression lines in the regenerated T0 plants or in the two independent events which produced T1 progeny. In addition to linking natural variation in tissue culture response to Wox2a, our data support the utility of Wox2a in enabling transformation of recalcitrant genotypes.     

A comparison between minirhizotron and monolith sampling methods for measuring root growth of maize (Zea mays L.)

Transparent plastic minirhizotron tubes have been used to evaluate spatial and temporal growth activities of plant root systems. Root number was estimated from video recordings of roots intersecting minirhizotron tubes and of washed roots extracted from monoliths of the same soil profiles at the physiological maturity stage of a maize (Zea mays L.) crop. Root length was measured by the line intercept (LI) and computer image processing (CIP) methods from the monolith samples.There was a slight significant correlation (r=0.28, p<0.005) between the number of roots measured by minirhizotron and root lengths measured by the LI method, however, no correlation was found with the CIP method. Using a single regression line, root number was underestimated by the minirhizotron method at depths between 0–7.6 cm. A correlation was found between root length estimated by LI and CIP. The slope of estimated RLD was significant with depth for these two methods. Root length density (RLD) measured by CIP showed a more erratic decline with distance from the plant row and soil surface than the LI method.     

Maturation of stamens and ovaries on cultured ear inflorescences of maize (Zea mays L.)

Observations were made on the maturation of stamens and ovaries from cultured maize (Zea mays L.) ear inflorescences. Immature ears (5.1–10.0 mm long) of maize were cultured in kinetin medium to study microsporogenesis and pollen maturation in developing stamens. Male spikelets developed on ears cultured in kinetin medium. Meiosis-I began by 7 days of culture in the developing anthers and the mature tri-nucleate pollen grains were developed by 20 days of culture. Further, kinetin was required in the culture medium for at least initial 5 days to obtain the microspores in differentiated stamens.To observe the embryosac formation in developed ovaries, ears were cultured in control, kinetin (10.1–15.0 mm long ears) medium, and kinetin + gibberellic acid (5.1–10.0 mm long ears) medium. Formation of embryosacs was noticed in the developed ovaries which were sampled after 20 days of culture. This differential flower development using two growth regulators provides an opportunity to uncover the biochemistry and physiology of micro- and mega-gametophyte development in maize.     

Phytotoxic effect of oxadiargyl on germination and early growth of sunflower (Helianthus annuus L.) and maize (Zea mays L.)

An attempt was made to assess the phytotoxicity of oxadiargyl on two crop plants, viz. sunflower and maize. The seed germination and early growth studies were carried out in hydroponic conditions with each variety of two crop plants, viz. sunflower (Morden) and maize (NAC-6004) supplemented with five concentrations of oxadiargyl. Seed germination and early growth of both the crop plants were significantly affected with increased concentration of oxadiargyl. The length of radicle and plumule was severely reduced along with increase in herbicidal concentration. Similarly, the calculated value of tolerance index and vigour index decreased drastically with increase in concentration of oxadiargyl. On the contrary, the per cent phytotoxicity also increased with increase in oxadiargyl concentration. Further, the pigment system of both the crop seedlings was severely affected by the herbicide oxadiargyl. These studies clearly indicate that oxadiargyl is phototoxic in nature.     

Incidence and severity of foliar diseases of sesame (Sesamum indicum L.) intercropped with maize (Zea mays L.)

This study evaluates the effects of different row arrangements on incidence and severity of Cercospora leaf spot (CLS) and Alternaria leaf blight (ALB) diseases and seed health of sesame intercropped with maize. Row arrangements were: sesame intercropped with maize in alternate pair of rows (2:2), two rows of sesame intercropped with one row of maize (2:1), sesame intercropped with maize in single alternate rows (1:1) with sole sesame as control. Intercropping maize with sesame reduced the incidence and severity of diseases. Sesame intercropped with maize in a (1:1) ration recorded a significantly lower number of infected leaves by CLS and ALB incidence than other row arrangements. ALB lesion number was between 17 and 20 in the (1:1) arrangement relative to 65–104 and 28–43 in the sole crop and other row arrangements, respectively. ALB lesion size was also reduced in the (1:1) than other row arrangements. Fungal infection of harvested sesame seeds was significantly reduced in the intercrop relative to the sole crop. CLS incidence was significant and negatively correlated with seed weight while defoliation was significant and positively correlated with ALB or CLS incidence. Rainfall was significant and positively correlated with CLS or ALB incidence while intercropping induced microclimatic effects that influenced disease incidence. Grain yield, weight of 1000-seed, number of capsules/plant and weight of seed/plant were significantly higher in the (1:1) row arrangement than the sole crop or other row arrangements. The study demonstrates that intercropping sesame with maize in a single alternate row (1:1) arrangement can be used to reduce foliar diseases of sesame.