Effects of soil and air drought on growth,plant water status and leaf gas exchange in three Mediterranean cedar species: <Emphasis Type="Italic">Cedrus atlantica</Emphasis>, <Emphasis Type="Italic">C. brevifolia</Emphasis> and <Emphasis Type="Italic">C. libani</Emphasis>

Three- and four-year-old potted, greenhouse-grown cedar seedlings were subjected to two different watering regimes: half received full water supply and the other half was submitted to moderate drought (50% of the full water supply). Height growth was the greatest for C. atlantica and the most-limited for C. brevifolia in the well-watered set. However, in the dry set, height growth was less affected by drought conditions for C. brevifolia than for C. atlantica. Cedrus libani gave intermediate results for both watering regimes. Moderate drought provoked a decrease in osmotic potential at full leaf turgor and a long-lasting osmotic adjustment. When irrigation was withheld completely to induce severe soil drying, gas exchange decreased and then stopped at predawn water potentials of −3.0 MPa for C. brevifolia, between −2.6 and −2.8 MPa for C. libani, and at −2.4 MPa for C. atlantica, irrespective of watering regime. For all species, the dry set showed lower net photosynthesis (A) and stomatal conductance (g _s) than the plants in the well-watered set. A and g _s responded to variations in atmospheric water-vapour pressure deficit (VPD). As VPD increased, A and g _s decreased, and this trend was proportionate to initial values at low VPD, but remained independent of previous watering treatments, plant water status or species. To conclude, C. brevifolia appears to be a species with limited growth potential but strong soil drought tolerance whereas C. atlantica has strong growth potential when an adequate water supply is available but is more sensitive to soil drought. C. libani shows an intermediate behaviour for growth and drought tolerance.     

Micro-colinearity between rice, <Emphasis Type="Italic">Brachypodium</Emphasis>, and <Emphasis Type="Italic">Triticum monococcum</Emphasis> at the wheat domestication locus <Emphasis Type="Italic">Q</Emphasis>

Brachypodium, a wild temperate grass with a small genome, was recently proposed as a new model organism for the large-genome grasses. In this study, we evaluated gene content and microcolinearity between diploid wheat (Triticum monococcum), Brachypodium sylvaticum, and rice at a local genomic region harboring the major wheat domestication gene Q. Gene density was much lower in T. monococcum (one per 41 kb) because of gene duplication and an abundance of transposable elements within intergenic regions as compared to B. sylvaticum (one per 14 kb) and rice (one per 10 kb). For the Q gene region, microcolinearity was more conserved between wheat and rice than between wheat and Brachypodium because B. sylvaticum contained two genes apparently not present within the orthologous regions of T. monococcum and rice. However, phylogenetic analysis of Q and leukotriene A-4 hydrolase-like gene orthologs, which were colinear among the three species, showed that Brachypodium is more closely related to wheat than rice, which agrees with previous studies. We conclude that Brachypodium will be a useful tool for gene discovery, comparative genomics, and the study of evolutionary relationships among the grasses but will not preclude the need to conduct large-scale genomics experiments in the Triticeae.     

Genomic analysis and expression pattern of <Emphasis Type="Italic">OsZIP1, OsZIP3</Emphasis>, and <Emphasis Type="Italic">OsZIP4</Emphasis> in two rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) genotypes with different zinc efficiency

The ZRT-and IRT-like proteins (ZIP) comprise a large family of transition metal transporters in plants that have diverse functions to transport zinc, iron, copper, etc. Here, we provided a complete overview of this gene family in rice (Oryza sativa L.). Based on the hidden Markov model and BLAST analysis, a total of 17 ZIP-coding genes were identified and further studied by semi-quantitative RT-PCR analysis. Sequence analysis revealed 17 putative genes distributed randomly on eight chromosomes. Although most of the predicted proteins had typical characteristics of the ZIP protein family, the extent of their sequence similarity varied considerably. The expression patterns of OsZIP1, OsZIP3, and OsZIP4, which encode Zn²⁺ transporters in rice, were studied in the Zn-efficient and Zn-inefficient rice genotypes (IR8192 and Erjiufeng) by semi-quantitative RT-PCR analysis of roots, shoots, and panicle from the plants grown under Zn deficiency and normal conditions. OsZIP1 was expressed only in the roots and very weakly if at all in the panicles, while the other two genes were expressed in all parts of plants under study. The Zn-deficient conditions up-regulated the expression of OsZIP1, OsZIP3, and OsZIP4 in the roots and that of OsZIP4 in the shoots of both genotypes, indicating that all these genes may participate in rice zinc nutrition. Furthermore, the expression of OsZIP3 and OsZIP4 was found to be much stronger in the roots of IR8192 than those of Erjiufeng, which suggests that these genes may contribute to high Zn efficiency in rice. The expression patterns and the roles of other OsZIPs are also discussed on the basis of the phylogenetic tree of ZIP proteins and RT-PCR analysis of the two rice genotypes with different zinc efficiency.     

A rice <Emphasis Type="Italic">YABBY</Emphasis> gene, <Emphasis Type="Italic">OsYABBY4</Emphasis>, preferentially expresses in developing vascular tissue

Developmental gene families have diversified during land plant evolution. The primary role of YABBY gene family is promoting abaxial fate in model eudicot, Arabidopsis thaliana. However recent results suggest that roles of YABBY genes are not conserved in the angiosperms. In this paper, a rice YABBY gene was isolated, and its expression patterns were analyzed in detail. Sequence characterization and phylogenetic analyses showed the gene is OsYABBY4, which is group-classified into FIL/YAB3 subfamily. Beta-glucuronidase reporter assay and in situ analysis consistently revealed that OsYABBY4 was expressed in the meristems and developing vascular tissue of rice, predominantly in the phloem tissue, suggesting that the function of the rice gene is different from those of its counterparts in eudicots. OsYABBY4 may have been recruited to regulate the development of vasculature in rice. However, transgenic Arabidopsis plants ectopically expressing OsYABBY4 behaved very like those over-expressing FIL or YAB3 with abaxialized lateral organs, suggesting the OsYABBY4 protein domain is conserved with its Arabidopsis counterparts in sequences. Our results also indicate that the functional diversification of OsYABBY4 may be associated with the divergent spatial-temporal expression patterns, and YABBY family members may have preserved different expression regulatory systems and functions during the evolution of different kinds of species.     

Fine mapping and candidate gene analysis of <Emphasis Type="Italic">hwh1</Emphasis> and <Emphasis Type="Italic">hwh2</Emphasis>, a set of complementary genes controlling hybrid breakdown in rice

Hybrid breakdown (HB), a phenomenon of reduced viability or fertility accompanied with retarded growth in hybrid progenies, often arises in the offspring of intersubspecific hybrids between indica and japonica in rice. We detected HB plants in F₈ recombinant inbred lines derived from the cross between an indica variety, Milyang 23, and a japonica variety, Tong 88-7. HB plants showed retarded growth, with fewer tillers and spikelets. Genetic analysis revealed that HB was controlled by the complementary action of two recessive genes, hwh1 and hwh2, originating from each of both parents, which were fine-mapped on the short arm of chromosome 2 and on the near centromere region of the long arm of chromosome 11, respectively. A comparison of the sequences of candidate genes among both parents and HB plants revealed that hwh1 encoded a putative glucose-methanol-choline oxidoreductase with one amino acid change compared to Hwh1 and that hwh2 probably encoded a putative hexose transporter with a six amino acid insertion compared to Hwh2. Investigation of the distribution of these alleles among 54 japonica and indica cultivars using candidate gene-based markers suggested that the two loci might be involved in developing reproductive barriers between two subspecies.     

The impact of <Emphasis Type="Italic">trans</Emphasis>-zeatin <Emphasis Type="Italic">O</Emphasis>-glucosyltransferase gene over-expression in tobacco on pigment content and gas exchange

The responses of tobacco plants over-expressing trans-zeatin O-glucosyltransferase gene under constitutive or senescence-inducible promoter (35S:ZOG1 and SAG12:ZOG1) and of wild type (WT) plants to water stress and subsequent rehydration were compared. In plants sufficiently supplied with water, both transgenics have higher net photosynthetic rate (P_N) in upper and middle leaves and higher stomatal conductance (g_s) in middle leaves than WT. Water use efficiency (WUE = P_N/E) was higher in both transgenics than in WT. During prolonged water stress, both P_N and E declined to a similar extent in both transgenics and WT plants. However, 7 d after rehydration P_N in SAG:ZOG (upper and middle leaves) and 35S:ZOG (upper leaves) was higher than that in WT plants. Increased content of endogenous CKs in 35S:ZOG plants did not prevent their response to ABA application and the results obtained did not support concept of CK antagonism of ABA-induced stomatal closure. The chlorophyll (Chl) a+b content was mostly higher in both transgenics than in WT. During water stress and subsequent rehydration it remained unchanged in upper leaves, decreased slightly in middle leaves only of WT, while rapidly in lower leaves. Total degradation of Chl, carotenoids and xanthophyll cycle pigments (XCP) was found under severe water stress in lower leaves. Carotenoid and XCP contents in middle and upper leaves mostly increased during development of water stress and decreased after rehydration. While β-carotene content was mostly higher in WT, neoxanthin content was higher in transgenics especially in 35S:ZOG under severe stress and after rehydration. The higher content of XCP and degree of their deepoxidation were usually found in upper and middle leaves than in lower leaves with exception of SAG:ZOG plants during mild water stress.     

Efficient authentic fine mapping of the rice blast resistance gene <Emphasis Type="Italic">Pik-h</Emphasis> in the <Emphasis Type="Italic">Pik</Emphasis> cluster,using new <Emphasis Type="Italic">Pik-h</Emphasis>-differentiating isolates

The Pik-h gene in rice confers resistance to several races of rice blast fungus (Magnaporthe oryzae), and has been classified as a member of the Pik cluster, one of the most resistance (R) gene-dense regions in the rice genome. However, the loss of a key mutant isolate has long made it difficult to differentiate Pik-h from other Pik group genes especially from Pik-m. We identified new natural isolates enabling the differentiation between Pik-h and Pik-m genes, and first confirmed the authenticity of the International Rice Research Institute (IRRI) “monogenic” line IRBLkh-K3, and then fine-mapped the Pik-h gene in the Pik cluster. Using 701 susceptible individuals among 3,060 siblings from a cross of IRBLkh-K3×CO39, the Pik-h region was delimited to 270 kb, the narrowest interval among the Pik group genes reported to date, in the cv. Nipponbare genome. Annotation of this genome region first revealed 6 NBS-LRR type R-gene analogs (RGAs), clustered within the central 120 kb, as possible counterparts of Pik-h and 6 other Pik group R genes. Interestingly, the Pik-h region and the cluster of RGAs were shown to be located 130 kb and 230 kb apart from Xa4 and Xa2 bacterial blight resistance genes, respectively, once classified as belonging to the Pik cluster. The closest recombination events were limited to the margins of the Pik-h region, and recombination was suppressed in the core interval with the RGA cluster. This fine-mapping, performed in a short time using an HEGS system, will facilitate utilization of the cluster’s genetic resources and help to elucidate the mechanism of evolution of R-genes. The presence of natural isolates also confirmed that evolution of Pik-h corresponds to pathogen evolution.     

Heterologous expression of <Emphasis Type="Italic">PDH47</Emphasis> confers drought tolerance in <Emphasis Type="Italic">indica</Emphasis> rice

Jerusalem artichoke (Helianthus tuberosus L.) cultivars are conserved in genebanks for use in breeding and horticultural research programs. Jerusalem artichoke collections are particularly vulnerable to environmental and biological threats because they are often maintained in the field. These field collections could be securely conserved in genebanks if improved cryopreservation methods were available. This work used four Jersualem artichoke cultivars (‘Shudi’, ‘M6’, ‘Stampede’, and ‘Relikt’) to improve upon an existing procedure. Four steps were optimized and the resulting procedure is as follows: preculture excised shoot tips (2–3 mm) in liquid MS medium supplemented with 0.4 M sucrose for 3 days, osmoprotect shoot tips in loading solution for 30 min, dehydrate with plant vitrification solution 2 for 15 min before rapid cooling in liquid nitrogen, store in liquid nitrogen, rapidly rewarm in MS liquid medium containing 1.2 M sucrose, and recover on MS medium supplemented with 0.1 mg L^?1 GA₃ for 3–5 days in the dark and then on the same medium for 4–6 weeks in the light (14 h light/10 h dark). After cryopreservation, Jerusalem artichoke cultivar ‘Shudi’ had the highest survival (93%) and regrowth (83%) percentages. Cultivars ‘M6’, ‘Stampede’, and ‘Relikt’ achieved survival and regrowth percentages ranging from 44 to 72%, and 37–53%, respectively. No genetic changes, as assessed by using simple sequence repeat markers, were detected in plants regenerated after LN exposure in Jerusalem artichoke cultivar ‘Shudi’. Differential scanning calorimetry analyses were used to investigate the thermal activities of the tissues during the cryopreservation process and it was determined that loading with 2.0 M sucrose and 0.4 M sucrose dehydrated the shoot tips prior to treatment with PVS2. Histological observations revealed that the optimized droplet vitrification protocol caused minimal cellular damage within the meristem cells of the shoot tips.     

Photosynthetic characteristics of <Emphasis Type="Italic">Hordeum,Triticum, Rumex</Emphasis>, and <Emphasis Type="Italic">Trifolium</Emphasis> species at contrasting altitudes

Photosynthetic characteristics were compared between plants of low altitude (LA) grown at LA (Palampur; 1 300 m) and at high altitude, HA (Kibber; 4 200 m), and plants naturally occurring at different altitudes (Palampur, 1 300 m; Palchan, 2 250 m; and Marhi, 3 250 m). Net photosynthetic rate (P _N) was not significantly different between altitudes. However, the slopes of the curve relating P _N to intercellular CO₂ concentration (C _i) were higher in plants at Palchan, Marhi, and Kibber compared to those at Palampur, indicating that plants had higher efficiency of carbon uptake (the initial slope of P _N/C _i curve is an indication) at HA. They had also higher stomatal conductance (g _s), transpiration rate, and lower water use efficiency at HA. g _s was insensitive to photosynthetic photon flux density (PPFD) for plants naturally occurring at Palampur, Palchan, and Marhi, whereas plants from LA grown at Palampur and Kibber responded linearly to increasing PPFD. Insensitivity of g _s to PPFD could be one of the adaptive features allowing wider altitudinal distribution of the plants.This research is supported by the Department of Biotechnology (DBT), Government of India vide grant number BT/PR/502/AGR/08/39/966-VI.     

Suppression by ABA of salicylic acid and lignin accumulation and the expression of multiple genes,in <Emphasis Type="Italic">Arabidopsis</Emphasis> infected with <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">tomato</Emphasis>

Abscisic acid (ABA) has been implicated in determining the outcome of interactions between many plants and their pathogens. We had previously shown that increased concentrations of ABA within leaves of Arabidopsis induced susceptibility towards an avirulent strain of Pseudomonas syringae pathovar (pv.) tomato. We now show that ABA induces susceptibility via suppression of the accumulation of components crucial for a resistance response. Lignin and salicylic acid concentrations in leaves were increased during a resistant interaction but reduced when plants were treated with ABA. The reduction in lignin and salicylic acid production was independent of the development of the hypersensitive response (HR), indicating that, in this host-pathogen system, HR is not required for resistance. Genome-wide gene expression analysis using microarrays showed that treatment with ABA suppressed the expression of many defence-related genes, including those important for phenylpropanoid biosynthesis and those encoding resistance-related proteins. Together, these results show that resistance induction in Arabidopsis to an avirulent strain of P. syringae pv. tomato is regulated by ABA. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Knockout of a starch synthase gene <Emphasis Type="Italic">OsSSIIIa</Emphasis>/<Emphasis Type="Italic">Flo5</Emphasis> causes white-core floury endosperm in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

To elucidate the role of SSIIIa during starch synthesis in rice (Oryza sativa L.) endosperm, we characterized null mutants of this gene, generated by T-DNA insertions. Scanning electron microscope (SEM) analysis revealed that the starch granules in these mutants are smaller and rounder compared with the wild type controls, and that the mutant endosperm is characterized by a loosely packed central portion exhibiting a floury-like phenotype. Hence, the OsSSIIIa (Oryza sativa SSIIIa) mutations are referred to as white-core floury endosperm 5-1 (flo5-1) and flo5-2. Based upon their X-ray diffraction patterns, the crystallinity of the starch in the flo5 mutant endosperm is decreased compared with wild type. Through determination of the chain-length distribution of the mutant endosperm starch, we found that flo5-1 and flo5-2 mutants have reduced the content of long chains with degree of polymerization (DP) 30 or greater compared with the controls. This suggests that OsSSIIIa/Flo5 plays an important role in generating relatively long chains in rice endosperm. In addition, DP 6 to 8 and DP 16 to 20 appeared to be reduced in endosperm starch of flo5-1 and flo5-2, whereas DP 9 to 15 and DP 22 to 29 were increased in these mutants. By the use of differential scanning calorimetry (DSC), the gelatinization temperatures of endosperm starch were found to be 1–5°C lower than those of the control. We propose a distinct role for OsSSIIIa/Flo5 and the coordinated action of other SS isoforms during starch synthesis in the seed endosperm of rice.     

<Emphasis Type="Italic">In vitro</Emphasis> stem elongation of stemless carline thistle

In vitro culture of stemless carline thistle was established using immature zygotic embryos. A satisfactory bud multiplication was achieved on MS medium supplemented with BAP (1 mg L^–1) and IAA (0.2 mg L^–1). Maximum rooting of buds was induced upon short cultivation (4 or 8 days) on an auxin-supplemented medium. Highest number of roots was obtained with NAA in the medium while the longest roots developed on the IAA-supplemented medium. Plantlets that subsequently developed were in rosette form if grown in light (16/8 h light to darkness photoperiod) and with elongated stems if raised in darkness. Light grown plantlets treated with GA₃ showed dose dependent stem increase in length, reaching maximum at the concentration of 10^–4 M. This was correlated with the length and the average number of internodes. If cultivated in the presence of ancymidol, dark grown plantlets showed reduced stem length. However, the inhibitory effect of the growth retardant on stem elongation was completely overcome by the addition of GA₃.     

Evaluation of physiological traits for improving drought tolerance in faba bean (<Emphasis Type="Italic">Vicia faba</Emphasis> L.)

Among grain legumes, faba bean is becoming increasingly popular in European agriculture due to recent economic and environmental interests. Faba bean can be a highly productive crop, but it is sensitive to drought stress and yields can vary considerably from season to season. Understanding the physiological basis of drought tolerance would indicate traits that can be used as indirect selection criteria for the development of cultivars adapted to drought conditions. To assess genotypic variation in physiological traits associated with drought tolerance in faba bean and to determine relationships among these attributes, two pot experiments were established in a growth chamber using genetic materials that had previously been screened for drought response in the field. Nine inbred lines of diverse genetic backgrounds were tested under adequate water supply and limited water conditions. The genotypes showed substantial variation in shoot dry matter, water use, stomatal conductance, leaf temperature, transpiration efficiency, carbon isotope discrimination (Δ¹³C), relative water content (RWC) and osmotic potential, determined at pre-flowering vegetative stage. Moisture deficits decreased water usage and consequently shoot dry matter production. RWC, osmotic potential, stomatal conductance and Δ¹³C were lower, whereas leaf temperature and transpiration efficiency were higher in stressed plants, probably due to restricted transpirational cooling induced by stomatal closure. Furthermore, differences in stomatal conductance, leaf temperature, Δ¹³C and transpiration efficiency characterized genotypes that were physiologically more adapted to water deficit conditions. Correlation analysis also showed relatively strong relationships among these variables under well watered conditions. The drought tolerant genotypes, ILB-938/2 and Melodie showed lower stomatal conductance associated with warmer leaves, whereas higher stomatal conductance and cooler leaves were observed in sensitive lines (332/2/91/015/1 and Aurora/1). The lower value of Δ¹³C coupled with higher transpiration efficiency in ILB-938/2, relative to sensitive lines (Aurora/1 and Condor/3), is indeed a desirable characteristic for water-limited environments. Finally, the results showed that stomatal conductance, leaf temperature and Δ¹³C are promising physiological indicators for drought tolerance in faba bean. These variables could be measured in pot-grown plants at adequate water supply and may serve as indirect selection criteria to pre-screen genotypes.     

Comparative genetics at the gene and chromosome levels between rice (<Emphasis Type="Italic">Oryza sativa</Emphasis>) and wildrice (<Emphasis Type="Italic">Zizania palustris</Emphasis>)

Using comparative genetics, genes, repetitive DNA sequences and chromosomes were studied in the Oryzeae in order to more fully exploit the rice genome sequence data. Of particular focus was Zizania palustris L., n = 15, commonly known as American wildrice. Previous work has shown that rice chromosomes 1, 4 and 9 are duplicated in wildrice. The Adh1 and Adh2 genes were sequenced and, based on phylogenetic analyses, found to be duplicated in wildrice. The majority of the sequence diversity in the Adh sequences was in intron 3, in which were found several MITE insertions. Cytological and molecular approaches were used to analyze the evolution of rDNA and centromeric repetitive sequences in the Oryzeae. In wildrice, copies of the 5S rDNA monomer were found at two loci on two different chromosomes near the centromeres, as in rice. One nucleolar organizer region (NOR) locus was found adjacent to the telomere, as in rice. RCS1, a middle repetitive sequence in rice, was present in all of the centromeres of wildrice. RCS2/CentO, the highly repetitive component of Oryza sativa L. centromeres, was conserved in eight of the Oryza species examined, but was not found in wildrice. Three other middle repetitive centromeric sequences (RCH1, RCH2/CentO and RCH3) were also examined and found to have variable evolutionary patterns between species of Oryza and Zizania.Communicated by B. Friebe     

Molecular characterization the <Emphasis Type="Italic">YABBY</Emphasis> gene family in <Emphasis Type="Italic">Oryza sativa</Emphasis> and expression analysis of <Emphasis Type="Italic">OsYABBY1</Emphasis>

Members of the YABBY gene family have a general role that promotes abaxial cell fate in a model eudicot, Arabidopsis thaliana. To understand the function of YABBY genes in monocots, we have isolated all YABBY genes in Oryza sativa (rice), and revealed the spatial and temporal expression pattern of one of these genes, OsYABBY1. In rice, eight YABBY genes constitute a small gene family and are classified into four groups according to sequence similarity, exon-intron structure, and organ-specific expression patterns. OsYABBY1 shows unique spatial expression patterns that have not previously been reported for other YABBY genes, so far. OsYABBY1 is expressed in putative precursor cells of both the mestome sheath in the large vascular bundle and the abaxial sclerenchyma in the leaves. In the flower, OsYABBY1 is specifically expressed in the palea and lemma from their inception, and is confined to several cell layers of these organs in the later developmental stages. The OsYABBY1-expressing domains are closely associated with cells that subsequently differentiate into sclerenchymatous cells. These findings suggest that the function of OsYABBY1 is involved in regulating the differentiation of a few specific cell types and is unrelated to polar regulation of lateral organ development.     

Changes in leaf photosynthesis of transgenic rice with silenced <Emphasis Type="Italic">OsBP-73</Emphasis> gene

In comparison with its wild type (WT), the transgenic (TG) rice with silenced OsBP-73 gene had significantly lower plant height, grain number per panicle, and leaf net photosynthetic rate (P _N). Also, the TG rice showed significantly lower chlorophyll (Chl), ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO), RuBPCO activase, and RuBP contents, photosystem 2 (PS2) photochemical efficiency (F_v/F_m and ΔF/F_m′), apparent quantum yield of carbon assimilation (Φ_c), carboxylation efficiency (CE), photosynthetic electron transport and photophosphorylation rates as well as sucrose phosphate synthase activity, but higher intercellular CO₂ concentration, sucrose, fructose, and glycerate 3-phosphate contents, and non-photochemical quenching of Chl fluorescence (NPQ). Thus the decreased P _N in the TG rice leaves is related to both RuBP carboxylation and RuBP regeneration limitations, and the latter is a predominant limitation to photosynthesis.