Characterization of two cDNAs for novel drought-inducible genes in the highly drought-tolerant cowpea

Ten cDNAs for drought-inducible genes were isolated using differential screening of a cDNA library prepared from 10-hr dehydrated cowpea plants,Vigna unguiculata (S. Iuchi, K. Yamaguchi-Shinozaki, T. Urao, T. Terao, K. Shinozaki; Plant Cell Physiology, 1996 in press). Two of the cDNA clones, designated CPRD12 and CPRD46, were sequenced and characterized. The CPRD12 and CPRD46 cDNAs encode putative proteins related to nonmetallo-short-chain alcohol dehydrogenase (CPRD12) and chloroplastic lipoxygenase (CPRD46). Northern blot analysis revealed that these genes are induced by high-salinity stress and exogenous abscisic acid, but not by cold stress. The CPRD46 gene is also responsive to heat stress and methyl jasmonate and salicylic acid. Genomic Southern blot analysis suggested that CPRD12 constitutes a small gene family, but that CPRD46 is a single copy gene. We discuss the possible functions of these two CPRD gene products under drought stress.     

Row spacing effects of N2-fixation,N-yield and soil N uptake of intercropped cowpea and maize

In the tropics, cowpea is often intercropped with maize. Little is known about the effect of the intercropped maize on N₂-fixation by cowpea or how intercropping affects nitrogen fertilizer use effiency or soil N-uptake of both crops. Cowpea and maize were grown as a monocrop at row spacings of 40, 50, 60, 80, and 120 cm and intercropped at row spacing of 40, 50, and 60 cm. Plots were fertilized with 50 kg N as (NH₄)₂SO₄; microplots within each plot received the same amount of¹⁵N-depleted (NH₄)₂SO₄. Using the¹⁵N-dilution method, the percentage of N derived from N₂-fixation by cowpea and the recovery of N-fertilizer and soil N-uptake was measured for both crops at 50 and 80 days after planting.Significant differences in yield and total N for cowpea and maize at both harvest periods were dependent on row spacing and cropping systems. Maize grown at the closer row spacing accumulated most of its N during the first 50 days after planting, whereas maize grown at the widest row spacing accumulated a significant portion of its N during the last 30 days before the final harvest, 80 days after planting.Overall, no significant differences in the percentage of N derived from N₂-fixation for monocropped or intercropped cowpea was observed and between 30 and 50% of its N was derived from N₂.At 50 DAP, fertilizer and soil N uptake was dependent on row spacing with maize grown at the narrowest row spacing having a higher fertilizer and soil N recovery than maize grown at wider spacings. At 50 and 80 DAP, intercropped maize/cowpea did not have a higher fertilizer and soil N uptake than monocropped cowpea or maize at the same row spacing. Monocropped maize and cowpea at the same row spacing took up about the same amount of fertilizer or soil N. When intercropped, maize took up twice as much soil and fertilizer N as cowpea. Apparently intercropped cowpea was not able to maintain its yield potential.Whereas significant differences in total N for maize was observed at 50 and 80 DAP, no significant differences in the atom %¹⁴N excess were observed. Therefore, in this study, the atom %¹⁴N excess of the reference crop was yield independent. Furthermore, the similarity in the atom %¹⁴N excess for intercropped and monocropped maize indicated that transfer of N from the legume to the non-legume was small or not detectable.     

Mineral N effects on cowpea and soybean crops in a Nigerian soil

Summary In a Nigerian soil depleted in available N, fertilizer-N enriched in¹⁵N was applied at 25 and 100 kg N/ha to crops of four cowpea and two soybean cultivars. Soil-N availability was estimated with three non-fixing crops, non-nodulating soybean, maize and celosia. With sequential harvests we examined the development of the fixing crops, as well as their nodulation profiles and acetylene reduction activities, and the patterns obtained were correlated with uptake of mineral-N. At low levels of mineral-N, excellent nodulation (up to 580 mg dry weight/plant) and very high acetylene reduction activities (up to 208 μmoles/plant/h) were recorded. Although fertilizer-N utilizations were low, 12% and 28% at 25 and 100 kg N/ha respectively, the lower application had a transient adverse effect on nodule development and the higher application had a long-term adverse effect on nodule formation, nodule development and acetylene reduction activity. Cowpea nodule mass reached maximum levels at early pod-fill except at 100 kg N/ha at which it continued to increase into late podfill. In contrast, soybean nodulation did not peak at any level of N but increased into late pod-fill.     

Cleavage polyembryony in maize

Summary Two types of cleavage polyembryony are described in the inbred line VIR 17 of maize. Suspensorial embryony was observed to occur spontaneously. Typical cleavage of the zygotic proembryo occurred spontaneously, but could also be induced by treating the developing caryopses with 2,4-Dichlorophenoxyacetic acid (2,4-D) on the second day after pollination. 2,4-D was active as a decorelative factor also evoking the expression of totipotency in individual proembryonal cells.     

Phosphorus use efficiency and nitrogen balance of cowpea breeding lines in a low P soil of the derived savanna zone in West Africa

Differences in growth, nodulation and arbuscular mycorrhizal fungi (AMF) root infection among recent cowpea breeding lines from IITA were examined at low and high P levels in pot (94 lines) and field experiments (43 lines) at Fashola in the derived savanna zone of Nigeria. Based on their growth performance, these lines were subdivided into 5 groups: (i) poor performance under low and high P conditions; (ii) good performance under low P and poor performance under high P; (iii) intermediate performance under high and low P; (iv) good performance under high and low P conditions; and (v) good performance under high P and poor performance under low P. About 42% of the breeding lines (18 out of 43 lines tested) had the same grouping for the field and pot experiments. Eight cowpea lines (4 P-responders and 4 non-P-responders) were selected from the first experiment for subsequent studies on the effect of P supply (0, 20, 40 and 60 kg P ha^-1) on P uptake, P use efficiency, dry matter production, N-fixation, AMF infection and N balance. Dry matter production, shoot/root ratio, total shoot N, and total N-fixed of the non-P-responder line, IT81D-715, were strongly related to P uptake efficiency. The P-responder IT81D-849 had a significant (95%) correlation between AMF and P-use efficiency. The cowpea lines fixed on average 22 kg N ha^-1, which was 70% of the plant total N. The N balance based on the difference between the amount of N₂ fixed and N exported through the harvest, ranged between −10.6 kg N ha^-1 and +7.7 kg N ha^-1. Based on its adaptability to grow in low P soils and overall positive N balance, the cowpea line IT81D-715 should be recommended for cultivation when P is the limiting factor. This revised version was published online in June 2006 with corrections to the Cover Date.     

Factors responsible for genotypic manganese tolerance in cowpea (Vigna unguiculata)

In experiments with 29 cowpea genotypes considerable variation in Mn tolerance could be found. Ranking according to Mn tolerance was almost the same in sand and water culture. Mn tolerance is not related to greater vigour or exclusion of Mn from uptake and translocation, but depends mainly on the internal tolerance to excess Mn especially in the leaf tissue.Growth depression by Mn excess is characterized by local accumulatiòn of Mn, deposition of Mn oxides, and typical macro-symptoms on the older leaves (brown spotsclorosisshedding of the leaves). Autoradiographic studies with⁵⁴Mn and extraction of the leaves with methanol and H₂O indicate a causal relationship between Mn tolerance and the more homogenous distribution of Mn in the tissue. In tolerant genotypes local accumulation and deposition of Mn is inhibited or retarded.Mn applied to the petioles of fully expanded leaves induces the same toxicity symptoms on the leaf blades as Mn absorbed by the roots. There is a good agreement between the rankings of the different genotypes for Mn tolerance according to the depression of shoot dry matter production by Mn excess in long term pot experiments and the appearance of toxicity symptoms after application of Mn to the petioles.The regulation of Mn tolerance at the leaf tissue level allows a quick and non-destructive screening of large numbers of genotypes for Mn tolerance.     

Analysis of pollen-tube growth in cultured maize silks

Summary In vitro pollen-tube growth in maize was studied using an in vitro pollination system. In the cut-silk method, ovaries with silks were placed on medium in vitro, whereafter the silk was cut and the upper part of the silk was pollinated. Pollen tubes were not able to bridge the space between the two silk parts. Even when silk parts were tightly connected, pollen tubes still were not able to pass the cut ends and reach the lower silk part. Pollen-tube growth rates and the direction of tube growth were not influenced by the presence or absence of an ovary. Prepollination did not have any influence on pollen-tube growth rate. Measurements of pollen-tube growth rate also showed that there was no population effect, i.e. growth rate was not stimulated by pollination with an excess of pollen grains. We found that the direction in which maize pollen grew was determined only by the positioning of the silk hairs.     

Developmental pattern formation of somatic embryos induced in cell suspension cultures of cowpea [Vigna unguiculata (L.) Walp]

The sequence of events in the functional body pattern formation during the somatic embryo development in cowpea suspensions is described under three heads. Early stages of somatic embryogenesis were characterized by both periclinal and anticlinal cell divisions. Differentiation of the protoderm cell layer by periclinal divisions marked the commencement of somatic embryogenesis. The most critical events appear to be the formation of apical meristems, establishment of apical-basal patterns of symmetry, and cellular organization in oblong-stage somatic embryo for the transition to torpedo and cotyledonary-stage somatic embryos. Two different stages of mature embryos showing distinct morphology, classified based on the number of cotyledons and their ability to convert into plantlets, were visualized. Repeated mitotic divisions of the sub-epidermal cell layers marked the induction of proembryogenic mass (PEM) in the embryogenic calli. The first division plane was periclinally-oriented, the second anticlinally-oriented, and the subsequent division planes appeared in any direction, leading to clusters of proembryogenic clumps. Differentiation of the protoderm layer marks the beginning of the structural differentiation in globular stage. Incipient procambium formation is the first sign of somatic embryo transition. Axial elongation of inner isodiametric cells of the globular somatic embryo followed by the change in the growth axis of the procambium is an important event in oblong-stage somatic embryo. Vacuolation in the ground meristem of torpedo-stage embryo begins the process of histodifferentiation. Three major embryonic tissue systems; shoot apical meristem, root apical meristem, and the differentiation of procambial strands, are visible in torpedo-stage somatic embryo. Monocotyledonary-stage somatic embryo induced both the shoot apical meristem and two leaf primordia compared to the ansiocotyledonary somatic embryo.     

Cowpea aphid performance and behaviour on two resistant cowpea lines

In a series of laboratory and climate chamber tests we compared the growth and behaviour of Aphis craccivora on one susceptible (ICV-1) and two aphid-resistant (ICV-11 and ICV-12) cowpea lines. The aphids' growth rates were much lower on the resistant cowpea lines than on the susceptible one, indicating strong antibiosis. In addition, the aphids invariably settled in higher numbers on the susceptible line than on either of the resistant. Compared to ICV-1, damaged leaves of the resistant line ICV-12 were settled upon to a higher degree than undamaged leaves, and leaf discs from the same line were even less resistant.On resistant lines individual aphids waited a significantly longer time before making their first test probe. Total probing time as well as the time preceding a decision to stay or leave was also longer.These results are discussed in relation to the possible mechanisms involved, and we also consider the effects of previous leaf feeding on the expression of resistance in the field.

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Résumé Au cours d'expériences au laboratoire et en chambres climatisées nous avons comparé la croissance et le comportement de A. craccivora sur une lignée sensible (ICV-1) et deux lignées résistantes (ICV-11 et ICV-12) de V. unguiculata. Les vitesses de croissance des pucerons ont été beaucoup plus lentes sur les lignées résistantes que sur la lignée sensible, ce qui révèle une forte antibiose. De plus, les pucerons atterrissent invariablement en plus grand nombre sur la variété sensible. Par comparaison avec ICV-1, les atterrissages sur lignée résistante ICV-12 étaient plus nombreux sur les feuilles endommagées que sur les feuilles intactes; les disques de feuilles de cette même lignée étaient encore moins résistants.Les pucerons ont séjourné individuellement un temps plus long sur les lignées résistantes avant de faire leur premier sondage. Le temps consacré aux sondages ainsi que le temps précédant de choix entre départ ou maintien sur la feuille étaient plus longs avec les lignées résistantes.Ces résultats ont été discutés en fonction des mécanismes impliqués. Nous avons aussi examiné les effets de la consommation antérieure sur les manifestations de la résistance dans la nature.

     

Effect of maize plants on colonisation of cowpea plants by bean flower thrips,Megalurothrips sjostedti

The effect of non-host maize plants on colonisation of cowpea byMegalurothrips sjostedti (Trybom) (Thysanoptera; Thripidae) was investigated. There were no differences in population density and activity ofM. sjostedti on sole cowpea crop and mixed cowpea/maize crop during the colonisation phase (i.e. 10–30 days after emergence of the plants. However, subsequentlyM. sjostedti numbers were lower in the mixed than in the sole crop, suggesting that maize did not interfere with colonisation of cowpea crop by thrips. In a choice situation, higher numbers ofM. sjostedti oriented towards, and settled on, sole cowpea plants than on those mixed with maize. Olfactory tests indicated that fewer thrips oriented towards a cowpea/maize mixed odour source. When equal numbers of thrips were introduced into the centre of sole- and mixed-cropped cowpea plots, the thrips became randomly distributed in each plot. Fewer thrips were recovered from the mixture than from the sole crop. It is concluded that, although non-host plant odours do not reduce thrips colonisation they interfere with host plant utilisation.     

Tillage effects on nitrogen uptake by maize from fine textured soils in the northwestern Corn Belt,USA

Summary Nitrogen (N) accumulation data from a replicated field study were fitted to a tanh (time) function and the derivate obtained to determine relative maximum rates of accumulation by maize. Both positive and negative effects of tillage on N accumulation rates were observed. Most of the N accumulation occurred over a 30-day period and time of N accumulation was not affected by tillage. Tilled profiles tend to contain greater NO₃–N, greater aeration, and lower moisture contents than untilled profiles, and these characteristics interact to affect plant N accumulation.     

Phosphorus effects on root growth and development in two maize genotypes

Soil phosphorus (P) availability is critical for the early growth and development of maize (Zea mays L.). Soil P also affects root morphological and physiological characteristics that are important for P uptake. The objective of this study was to evaluate the effects of P on seedling root growth and development of two maize genotypes differing in root system plasticity. Two maize genotypes, CM37 (high plasticity) and W153R (low plasticity), were selected based on a preliminary study. Maize plants were evaluated at six vegetative stages of development for three soil P treatments (0, 45, and 300 mg kg^-1). Seedlings were grown in a controlled environment using a soil with low native P, Maddock sandy loam (sandy, mixed Udorthentic Haploborolls). The addition of P decreased the time to reach a given growth stage and increased the relative growth rate of roots to a greater degree in CM37 than in W153R. The effects of P on shoot dry weight and root surface area during the V4–V6 growth period appeared to be related to the effects of P on development and relative growth rates during the V1–V3 growth period. Evaluation of the time course of phenotypic change is an important consideration when developing adapted genotypes for specific environments.     

Mycorrhizal colonization in cowpea is host dependent and heritable

Cowpea genotypes (101) of diverse origin were evaluated for their endomycorrhizal colonization under natural field conditions in an alfisol soil. There was considerable variation in colonization ranging from 0 to 28.6%. The genetic coefficient of variability was high (55.9), heritability moderate (46.2) with high genetic advance (78.3) revealing that mycorrhizal colonization is not only host dependent but heritable.     

Overexpression of <Emphasis Type="Italic">Zm401</Emphasis>, an mRNA-like RNA,has distinct effects on pollen development in maize

We previously identified a 0.7 Kb cDNA fragment of Zm401, a novel pollen-specific gene in maize (Zea mays). However, little information is known about the function of Zm401 in pollen development. The full-length of Zm401 cDNA was amplified by 5′ RACE and 3′ RACE and both sequence analysis and in vitro translation of Zm401 showed that it belonged to an mRNA-like non-coding gene. To analyze its possible biological roles in pollen development, the Zm401 cDNA was overexpressed in transgenic maize under the control of a pollen specific promoter Zm13 or a CaMV 35S promoter. RT-PCR and RNA gel blot analysis indicated that the expression level of Zm401 in leaves and anthers of transgenic plants was much higher than that of non-transformants. Compared with the non-transformed maize, transgenic maize showed distinct phenotypes, such as abnormal tassels and degenerate anthers. The histological observation showed that the development of pollen grains and anthers in transgenic plants were abnormal. These abnormalities include delayed degradation of tapetum, asynchronous fusion of pollen sacs, and aborted pollen grain development. Furthermore, the pollen viability in six transgenic plants ranged from 1.24% to 6.63%. The reduced pollen viability cosegregated with the transgene in a selfed progeny. These results suggest that Zm401 is involved in the regulation of pollen development. This article demonstrated Zm401, as a non-coding RNA, plays an essential role in pollen development. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The variations of NPK of cowpea (Vigna unguiculata (L) Walp) leaves with tillage,plant maturity and flooding on a clay soil

Summary The NPK concentrations in cowpea leaves (cv California Blackeye No. 5) with stage of plant development did not vary significantly with tillage treatment but several features of the concentration of these different ions with age, stage of plant development, incidence of flooding and soil water content are significant.In the major rainy season when soil water content was adequate and seldom above field capacity, there was an almost linear increase in the concentration of N and P in the leaves up to 56 days after planting which coincided with fruit development. This suggested that the accumulation of N and P do take place after flowering which occurred 39 days after planting. The pattern of accumulation of N and P in the minor rainy season suggested that flooding and the occurrence of prolonged soil moisture contents above field capacity suppressed the accumulation of these two nutrients. Potassium accumulation was high in the early stages of cowpea growth and declined with age of the crop. Flooding and high soil moisture contents seem to have had little or no effect on potassium accumulation in the leaves. The pattern of accumulation of N and P suggests that the supply of these nutrients to the cowpea plant must remain high up to fruit development and that additional N and P would need to be added to the soil after a flooding event or when the soil moisture content is above field capacity for prolonged periods.     

Inhibition of phosphoenolpyruvate carboxylase from maize by 2-phosphoglycollate

The phosphoenolpyruvate carboxylase from maize leaf was strongly inhibited by 2-phosphoglycollate. The pH of the reaction did not influence the extent of inhibition by 2-phosphoglycollate. The kinetic analysis of the inhibition data by Lineweaver-Burk method showed that 2-phosphoglycollate inhibition was competitive with respect to phosphoenolpyruvate. The secondary plot of the data showed nonlinearity indicating that there may be two 2-phosphoglycollate binding sites with Ki values of 0.4 mM and 0.16 mM. The biphasic nature of the inhibition was also evident when the data were plotted using the method of Dixon. 2-phosphoglycollate inhibition was uncompetitive with respect to Mg²⁺ suggestting that it binds only to enzyme-Mg²⁺ complex.     

Inheritance of resistance to the cowpea aphid in cowpea

Summary Inheritance of resistance to cowpea aphid, Aphis craccivora Koch, in three resistant cultivars of cowpea, Vigna unguiculata (L.) Walp, was studied. The parents, F₁ and F₂ population were grown in an insect-proof screenhouse. Each 3-day-old seedling was infested with 10 apterous adult aphids. Seedling reaction was recorded when the susceptible check was killed. The segregation data revealed that the resistance of ICV11 and TVU310 is governed by single dominant genes. All the F₂ seedlings of the cross ICV10xTVU310 were resistant, indicating that they have the same gene for resistance. However, the F₂ populations from the crosses ICV10xICV11 and ICV11xTVU310 segregated in a ratio of 151, indicating that the dominant genes in ICV11 and TVU310 are non-allelic and independent of each other. The resistance gene of ICV10 and TVU310 is designated as Ac₁ and that of ICV11 as Ac₂.     

Partitioning of biologically fixed nitrogen in cowpea during pod development

Two days after exposure of roots to¹⁵N labeled N₂, partitioning of biologically fixed N into leaves, stems, peduncles, pods, roots and nodules was measured in the early pod development stage of cowpea (Vigna unguiculata (L.). The experimental objective was to determine the quantity of biologically fixed N that is incorporated into vegetative tissue before being mobilized to pods. For the three varieties of cowpea included in the experiment a maximum of 50% of the N, biologically fixed two days earlier, was contained in the pods. The remaining N was distributed throughout the vegetative portion of the plant with at least 30% in stems and leaves which indicates that much of the newly fixed N must cycle through a N pool in these tissues before reaching the pods.     

Influence of 2,4-D,TIBA and 3,5-D on the growth response of cultured maize embryos

Induction of embryogenic calli from immature zygotic embryos of maize requires the presence of 2,4-D or similar auxin-like growth regulators in the culture medium. Pulse-chase experiments with 2,4-D, using various concentrations of 2,4-D in the induction medium were tested in relation to induction of callus in the embryogenic inbred line A188 and the non-embryogenic inbred A632. Interactions of 2,4-D, 3,5-D and the auxin transport inhibitor TIBA were also studied. Pulse-chase experiments showed that exposure to 2,4-D influenced the culture response from 0.5 h onwards. After a pulse of 0.5 h, shoot and root elongation of the embryo was stimulated. A pulse of 16 h or longer induced outgrowths and callus formation at the basal side of the scutellum. Pulses of 7 days and longer resulted in the induction of friable embryogenic Type II callus in A188. Embryos were cultured at 2,4-D concentrations ranging from 0.002 to 2000 mg l⁻¹ and optimal concentration for the induction of embryogenic callus in A188 was 2 mg l⁻¹. At lower concentrations there was a transition between callus formation and germination; at increasing concentrations, callus induction was reduced and finally growth responses became blocked. When TIBA was added to medium without 2,4-D, root elongation decreased in a dose-dependent way suggesting the need of polar transport of endogenous auxins for root elongation. When added to medium with 2,4-D, TIBA caused suppression of callus formation, again pointing to the necessity of polar transport of 2,4-D. In combination with 2,4-D, cultures with 3,5-D resembled cultures at lower 2,4-D concentrations, pointing to a competitive interaction between 3,5-D and 2,4-D. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of 1.10-phenanthroline, a photodynamic herbicide on the development and structure of maize chloroplasts

Effect of low (5 mmol·dm⁻³) and high (10 or 20 mmol·dm⁻³) doses of 1.10-phenanthroline (Phe), a photodynamic herbicide, on the development of chloroplasts in etiolated and subsequently illuminated maize seedlings and on the structure of already developed chloroplasts of green maize seedlings was examined. Etiolated and then irradiated plants were resistant to 5 mmol·dm⁻³ of Phe with respect to morphology, however Phe caused inhibition of greening and of grana formation. Higher Phe concentrations followed by exposure to light caused not only total inhibition of greening but also dilation of thylakoids, swelling of chloroplasts, and finally total destruction of chloroplast structure. Application of Phe in the same concentrations to green plants revealed that they were resistant to low dose of Phe with respect to morphology and structure of chloroplasts, however 10 and 20 mmol·dm⁻³ Phe and illumination caused the loss of turgor of treated plants and other photooxidative damages seen at the ultrastructural level. We concluded that maize, as representant of monocotyledonous plants, is resistant to low (5 mmol·dm⁻³) Phe concentration. Higher (10 or 20 mmol·dm⁻³) concentrations, used to determine the site of damage and mode of action of Phe on the level of cell revealed that action of photodynamic herbicides is based on standard photoinhibition mechanism and also probably on their chelating properties.