An Arabidopsis homolog of yeast ATG6/VPS30 is essential for pollen germination

Yeast (Saccharomyces cerevisiae) Atg6/Vps30 is required for autophagy and the sorting of vacuolar hydrolases, such as carboxypeptidase Y. In higher eukaryotes, however, roles for ATG6/VPS30 homologs in vesicle sorting have remained obscure. Here, we show that AtATG6, an Arabidopsis (Arabidopsis thaliana) homolog of yeast ATG6/VPS30, restored both autophagy and vacuolar sorting of carboxypeptidase Y in a yeast atg6/vps30 mutant. In Arabidopsis cells, green fluorescent protein-AtAtg6 protein localized to punctate structures and colocalized with AtAtg8, a marker protein of the preautophagosomal structure. Disruption of AtATG6 by T-DNA insertion resulted in male sterility that was confirmed by reciprocal crossing experiments. Microscopic analyses of AtATG6 heterozygous plants (AtATG6/atatg6) crossed with the quartet mutant revealed that AtATG6-deficient pollen developed normally, but did not germinate. Because other atatg mutants are fertile, AtAtg6 likely mediates pollen germination in a manner independent of autophagy. We propose that Arabidopsis Atg6/Vps30 functions not only in autophagy, but also plays a pivotal role in pollen germination.     

The autophagy-associated Atg8 gene family operates both under favourable growth conditions and under starvation stresses in Arabidopsis plants

Arabidopsis plants possess a family of nine AtAtg8 gene homologues of the yeast autophagy-associated Apg8/Aut7 gene. To gain insight into how these genes function in plants, first, the expression patterns of five AtAtg8 homologues were analysed in young Arabidopsis plants grown under favourable growth conditions or following exposure to prolonged darkness or sugar starvation. Promoters, plus the entire coding regions (exons and introns) of the AtAtg8 genes, were fused to the beta-glucuronidase reporter gene and transformed into Arabidopsis plants. In all plants, grown under favourable growth conditions, beta-glucuronidase staining was much more significant in roots than in shoots. Different genes showed distinct spatial and temporal expression patterns in roots. In some transgenic plants, beta-glucuronidase staining in leaves was induced by prolonged darkness or sugar starvation. Next, Arabidopsis plants were transformed with chimeric gene-encoding Atg8f protein fused to N-terminal green fluorescent protein and C-terminal haemagglutinin epitope tags. Analysis of these plants showed that, under favourable growth conditions, the Atg8f protein is efficiently processed and is localized to autophagosome-resembling structures, both in the cytosol and in the central vacuole, in a similar manner to its processing and localization under starvation stresses. Moreover, treatment with a cocktail of proteasome inhibitors did not prevent the turnover of this protein, implying that its turnover takes place in the vacuoles, as occurs in yeasts. The results suggest that, in plants, the cellular processes involving the Atg8 genes function efficiently in young, non-senescing tissues, both under favourable growth conditions and under starvation stresses.     

Autophagy-related gene, TdAtg8, in wild emmer wheat plays a role in drought and osmotic stress response

An autophagy-related gene Atg8 was cloned for the first time from wild emmer wheat, named as TdAtg8, and its role on autophagy under abiotic stress conditions was investigated. Examination of TdAtg8 expression patterns indicated that Atg8 expression was strongly upregulated under drought stress, especially in the roots when compared to leaves. LysoTracker(?) red marker, utilized to observe autophagosomes, revealed that autophagy is constitutively active in Triticum dicoccoides. Moreover, autophagy was determined to be induced in plants exposed to osmotic stress when compared to plants grown under normal conditions. Functional studies were executed in yeast to confirm that the TdATG8 protein is functional, and showed that the TdAtg8 gene complements the atg8?::kan MX yeast mutant strain grown under nitrogen deficiency. For further functional analysis, TdATG8 protein was expressed in yeast and analyzed using Western immunoblotting. Atg8-silenced plants were exposed to drought stress and chlorophyll and malondialdehyde (MDA) content measurements demonstrated that Atg8 plays a key role on drought stress tolerance. In addition, Atg8-silenced plants exposed to osmotic stress were found to have decreased Atg8 expression level in comparison to controls. Hence, Atg8 is a positive regulator in osmotic and drought stress response.     

AtATG genes, homologs of yeast autophagy genes, are involved in constitutive autophagy in Arabidopsis root tip cells

In Arabidopsis root tips cultured in medium containing sufficient nutrients and the membrane-permeable protease inhibitor E-64d, parts of the cytoplasm accumulated in the vacuoles of the cells from the meristematic zone to the elongation zone. Also in barley root tips treated with E-64, parts of the cytoplasm accumulated in autolysosomes and pre-existing central vacuoles. These results suggest that vacuolar and/or lysosomal autophagy occurs constitutively in these regions of cells. 3-Methyladenine, an inhibitor of autophagy, inhibited the accumulation of such inclusions in Arabidopsis root tip cells. Such inclusions were also not observed in root tips prepared from Arabidopsis T-DNA mutants in which AtATG2 or AtATG5, an Arabidopsis homolog of yeast ATG genes essential for autophagy, is disrupted. In contrast, an atatg9 mutant, in which another homolog of ATG is disrupted, accumulated a significant number of vacuolar inclusions in the presence of E-64d. These results suggest that both AtAtg2 and AtAtg5 proteins are essential for autophagy whereas AtAtg9 protein contributes to, but is not essential for, autophagy in Arabidopsis root tip cells. Autophagy that is sensitive to 3-methyladenine and dependent on Atg proteins constitutively occurs in the root tip cells of Arabidopsis.     

Salt stress signals shape the plant root

Plants use different strategies to deal with high soil salinity. One strategy is activation of pathways that allow the plant to export or compartmentalise salt. Relying on their phenotypic plasticity, plants can also adjust their root system architecture (RSA) and the direction of root growth to avoid locally high salt concentrations. Here, we highlight RSA responses to salt and osmotic stress and the underlying mechanisms. A model is presented that describes how salinity affects auxin distribution in the root. Possible intracellular signalling pathways linking salinity to root development and direction of root growth are discussed. These involve perception of high cytosolic Na+ concentrations in the root, activation of lipid signalling and protein kinase activity and modulation of endocytic pathways.     

Cytokinin action in response to abiotic and biotic stresses in plants

The phytohormone cytokinin was originally discovered as a regulator of cell division. Later, it was described to be involved in regulating numerous processes in plant growth and development including meristem activity, tissue patterning, and organ size. More recently, diverse functions for cytokinin in the response to abiotic and biotic stresses have been reported. Cytokinin is required for the defence against high light stress and to protect plants from a novel type of abiotic stress caused by an altered photoperiod. Additionally, cytokinin has a role in the response to temperature, drought, osmotic, salt, and nutrient stress. Similarly, the full response to certain plant pathogens and herbivores requires a functional cytokinin signalling pathway. Conversely, different types of stress impact cytokinin homeostasis. The diverse functions of cytokinin in responses to stress and crosstalk with other hormones are described. Its emerging roles as a priming agent and as a regulator of growth‐defence trade‐offs are discussed.     

Cytokinin-deficient transgenic Arabidopsis plants show multiple developmental alterations indicating opposite functions of cytokinins in the regulation of shoot and root meristem activity

Cytokinins are hormones that regulate cell division and development. As a result of a lack of specific mutants and biochemical tools, it has not been possible to study the consequences of cytokinin deficiency. Cytokinin-deficient plants are expected to yield information about processes in which cytokinins are limiting and that, therefore, they might regulate. We have engineered transgenic Arabidopsis plants that overexpress individually six different members of the cytokinin oxidase/dehydrogenase (AtCKX) gene family and have undertaken a detailed phenotypic analysis. Transgenic plants had increased cytokinin breakdown (30 to 45% of wild-type cytokinin content) and reduced expression of the cytokinin reporter gene ARR5:GUS (beta-glucuronidase). Cytokinin deficiency resulted in diminished activity of the vegetative and floral shoot apical meristems and leaf primordia, indicating an absolute requirement for the hormone. By contrast, cytokinins are negative regulators of root growth and lateral root formation. We show that the increased growth of the primary root is linked to an enhanced meristematic cell number, suggesting that cytokinins control the exit of cells from the root meristem. Different AtCKX-green fluorescent protein fusion proteins were localized to the vacuoles or the endoplasmic reticulum and possibly to the extracellular space, indicating that subcellular compartmentation plays an important role in cytokinin biology. Analyses of promoter:GUS fusion genes showed differential expression of AtCKX genes during plant development, the activity being confined predominantly to zones of active growth. Our results are consistent with the hypothesis that cytokinins have central, but opposite, regulatory functions in root and shoot meristems and indicate that a fine-tuned control of catabolism plays an important role in ensuring the proper regulation of cytokinin functions.     

The putative plasma membrane Na(+)/H(+) antiporter SOS1 controls long-distance Na(+) transport in plants

The salt tolerance locus SOS1 from Arabidopsis has been shown to encode a putative plasma membrane Na(+)/H(+) antiporter. In this study, we examined the tissue-specific pattern of gene expression as well as the Na(+) transport activity and subcellular localization of SOS1. When expressed in a yeast mutant deficient in endogenous Na(+) transporters, SOS1 was able to reduce Na(+) accumulation and improve salt tolerance of the mutant cells. Confocal imaging of a SOS1-green fluorescent protein fusion protein in transgenic Arabidopsis plants indicated that SOS1 is localized in the plasma membrane. Analysis of SOS1 promoter-beta-glucuronidase transgenic Arabidopsis plants revealed preferential expression of SOS1 in epidermal cells at the root tip and in parenchyma cells at the xylem/symplast boundary of roots, stems, and leaves. Under mild salt stress (25 mM NaCl), sos1 mutant shoot accumulated less Na(+) than did the wild-type shoot. However, under severe salt stress (100 mM NaCl), sos1 mutant plants accumulated more Na(+) than did the wild type. There also was greater Na(+) content in the xylem sap of sos1 mutant plants exposed to 100 mM NaCl. These results suggest that SOS1 is critical for controlling long-distance Na(+) transport from root to shoot. We present a model in which SOS1 functions in retrieving Na(+) from the xylem stream under severe salt stress, whereas under mild salt stress it may function in loading Na(+) into the xylem.     

胡杨细胞和组织结构与其耐盐性关系的研究

The characteristics of the cell and organ structures of Populus euphratica Oliv. in relation to salt and osmotic tolerance were compared with those of P. tomentosa Cart. in vitro under the electron and light microscopic observation. P. euphratica exhibited characteristic structure which was associated with salt stress. It had well-developed epidermis and exodermis in the root tip and poorly developed conducting tissue in leaf. Root hairs were formed closer to the root tips. AsP. euphratica were stressed with salt and PEG, more abundance of chondriosomes and plastids in the cytoplasm and more containing substance in the plastid were observed and the osmophilic substance was obviously displayed in the cytoplasm and in the posterior margin of the vacuole. The filamentous structure, bigger nucleus and nucleolus were visualized in the stressed suspension-cultured cells of P. euphratica. The meristemic cells in the root tip of P. euphratica could maintain their structure when the plant was subjected to 8 g/L NaCl stress. It was also demonstrated that the cell wall and plasmalemma of P. euphratica were tightly combined as a dentate form, explaining why the cell could endure severe salt or osmotic stress and resist to plasmolysis indicating that P. euphratica possesses a solid structure base as a defense to salt stress.     

Auxin–cytokinin interplay shapes root functionality under low-temperature stress

Low-temperature stress alters root system architecture. In particular, changes in the levels and response to auxin and cytokinin determine the fate of root architecture and function under stress because of their vital roles in regulating root cell division, differentiation, and elongation. An intricate nexus of genes encoding components of auxin and cytokinin biosynthesis, signaling, and transport components operate to counteract stress and facilitate optimum development. We review the role of auxin transport and signaling and its regulation by cytokinin during root development and stem cell maintenance under low-temperature stress. We highlight intricate mechanisms operating in root stem cells to minimize DNA damage by altering phytohormone levels, and discuss a working model for cytokinin in low-temperatures stress response.     

Constitutive autophagy in plant root cells

In previous studies, using a membrane-permeable protease inhibitor, E-64d, we showed that autophagy occurs constitutively in the root cells of barley and Arabidopsis. In the present study, a fusion protein composed of the autophagy-related protein AtAtg8 and green fluorescent protein (GFP) was expressed in Arabidopsis to visualize autophagosomes. We first confirmed the presence of autophagosomes with GFP fluorescence in the root cells of seedlings grown on a nutrient-sufficient medium. The number of autophagosomes changed as the root cells grew and differentiated. In cells near the apical meristem, autophagosomes were scarcely found. However, a small but significant number of autophagosomes existed in the elongation zone. More autophagosomes were found in the differentiation zone where cell growth ceases but the cells start to form root hair. In addition, we confirmed that autophagy is activated under starvation conditions in Arabidopsis root cells. When the root tips were cultured in a sucrose-free medium, the number of autophagosomes increased in the elongation and differentiation zones, and a significant number of autophagosomes appeared in cells near the apical meristem. The results suggest that autophagy in plant root cells is involved not only in nutrient recycling under nutrient-limiting conditions but also in cell growth and root hair formation.     

荒漠绿洲过渡带一年生草本植物对干旱胁迫的响应

选取河西走廊荒漠绿洲过渡带典型一年生草本植物雾冰藜（Bassia dasyphylla）、虎尾草（Chloris virgata）和狗尾草（Setaria viridis）为研究对象,设置5个水分梯度（正常水分（CK）,轻度干旱（5d）、中度干旱（10d）、重度干旱（15d）,重度干旱（15d）复水）,分析了3种一年生草本植物生理和形态等性状对干旱胁迫的响应。结果表明：一年生草本植物可以通过生理反应（渗透调节）适应轻度和中度干旱胁迫,而通过个体形态来适应重度干旱胁迫。在轻度和中度干旱处理下,一年生草本植物通过调控叶片渗透调节物质脯氨酸、可溶性蛋白和可溶性糖维持叶片渗透压,提高保水能力,叶绿素含量增加,使丙二醛含量维持在较低的水平,同时,根系活力增强,有效促进了根系水分吸收;而在重度胁迫下,渗透调节物质作用降低,丙二醛含量迅速增加,导致可溶性蛋白含量下降,叶绿素分解加速,植物生长受到抑制,在有限的生物量下,一年生草本植物主要通过根系伸长、根长与茎长的比增加和减小茎长来适应重度干旱胁迫,最终导致了种子百粒重和结种数量下降。     

Na~ and Water Uptake in Relation to the Radial Reflection Coefficient of Root in Arrowleaf Saltbush Under Salt Stress

The response of halophyte arrowleaf saltbush(Atriplex triangularis Willd)plants to a gradient of salt stress were investigatedwith hydroponically cultured seedlings.Under salt stress,both the Na~ uptake into root xylem and negative pressures inxylem vessels increased with the elevation of salinity(up to 500 mol/m~3)in the root environment.However,the increment innegative pressures in root xylem far from matches the decrease in the osmotic potential of the root bathing solutions,evenwhen the osmotic potential of xylem sap is taken into consideration.The total water potential of xylem sap in arrowleafsaltbush roots was close to the osmotic potential of root bathing solutions when the salt stress was low,but a progressivelyincreased gap between the water potential of xylem sap and the osmotic potential of root bathing solutions was observedwhen the salinity in the root environment was enhanced.The maximum gap was 1.4 MPa at a salinity level of 500 mol/m~3without apparent dehydration of the tested plants.This discrepancy could not be explained with the current theories inplant physiology.The radial reflection coefficient of root in arrowleaf saltbush decreased with the enhanced salt stress wasand accompanied by an increase in the Na~ uptake into xylem sap.However,the relative Na~ in xylem exudates based onthe corresponding NaCl concentration in the root bathing solutions showed a tendency of decrease.The results showedthat the reduction in the radial reflection coefficient of roots in the arrowleaf saltbush did not lead to a mass influx of NaClinto xylem when the radial reflection coefficient of the root was considerably small;and that arrowleaf saltbush could usesmall xylem pressures to counterbalance the salt stresses,either with the uptake of large amounts of salt,or with thedevelopment of xylem pressures dangerously negative.This strategy could be one of the mechanisms behind the highresistance of arrowleaf saltbush plants to salt stress.     

Expression of three <Emphasis Type="Italic">Arabidopsis</Emphasis> cytokinin oxidase/dehydrogenase promoter::GUS chimeric constructs in tobacco: response to developmental and biotic factors

Expression patterns of three Arabidopsis thaliana cytokinin oxidase/dehydrogenase promoter::GUS reporter fusions were investigated in tobacco plants. While cytokinin oxidase/dehydrogenase promoter 2 showed no expression in tobacco, the cytokinin oxidase/dehydrogenase promoters 3 and 4 were active in various tissues throughout development of the tobacco. Recently, the 1452 bp promoter region of AtCKX3 was reported as almost inactive in Arabidopsis. In contrast, the 1627 bp DNA fragment preceding the AtCKX3 coding region drove expression of the reporter GUS gene in various tobacco tissues. The promoter was mainly expressed in tobacco leaves and roots during early stages of development but also later in young flower buds as well as in pollen grains. The construct was particularly active before (hypocotyl region) and during (vascular system) lateral root initiation, supporting the idea of an inhibitory role of active cytokinins in the process of root initiation. The cytokinin oxidase/dehydrogenase promoter 4::GUS fusion in tobacco was shown to share some common (but weaker) expression patterns with promoter 3, namely in the leaves and pollen, but also conferred specific expression in tobacco root cap cells and trichomes. In addition, the response of cytokinin oxidase/dehydrogenase promoter::GUS reporter fusions to infection with the leafy gall-forming bacteria Rhodococcus fascians was examined. While an avirulent strain of R. fascians did not induce expression of any of the cytokinin oxidase/dehydrogenase promoters, the cytokinin oxidase/dehydrogenase promoter 3::GUS fusion was specifically induced at the site of infection when plants were challenged with a virulent strain of R. fascians, providing a possible explanation for the lack of significantly elevated cytokinin concentrations in tissues infected with virulent strains of R. fascians.This revised version was published online in August 2005 with some black and white figures replaced by coloured figures.     

The soybean root‐specific protein kinase GmWNK1 regulates stress‐responsive ABA signaling on the root system architecture

In humans, members of the WNK protein kinase family are osmosensitive regulators of cell volume homeostasis and epithelial ion transport, and mutation of these proteins causes a rare inherited form of hypertension due to increased renal NaCl re‐absorption. A related class of kinases was recently discovered in plants, but their functions are largely unknown. We have identified a root‐specific WNK kinase homolog, GmWNK1, in soybean (Glycine max). GmWNK1 expression was detected in the root, specifically in root cells associated with lateral root formation, and was down‐regulated by abscisic acid (ABA), as well as by mannitol, sucrose, polyethylene glycol and NaCl. In vitro and in vivo experiments showed that GmWNK1 interacts with another soybean protein, GmCYP707A1, which is a key ABA 8′‐hydroxylase that functions in ABA catabolism. Furthermore, 35S‐GmWNK1 transgenic soybean plants had reduced lateral root number and length compared with wild‐type, suggesting a role of GmWNK1 in the regulation of root system architecture. We propose that GmWNK1 functions to fine‐tune ABA‐dependent ABA homeostasis, thereby mediating the regulation of the root system architecture by ABA and osmotic signals. The study has revealed a new function of a plant WNK1 gene from the important staple crop soybean, and has identified a new component of a regulatory pathway that is involved not only in ABA signaling, but also in the repression of lateral root formation by an ABA‐dependent mechanism distinct from known ABA signaling pathways.     

Overexpression of AeNHX1, a root-specific vacuolar Na+/H+ antiporter from Agropyron elongatum, confers salt tolerance to Arabidopsis and Festuca plants

Agropyron elongatum, a species in grass family, has a strong tolerance to salt stress. To study the molecular mechanism of Agropyron elongatum in salt tolerance, we isolated a homolog of Na⁺/H⁺ antiporters from the root tissues of Agropyron plants. Sequence analysis revealed that this gene encodes a putative vacuolar Na⁺/H⁺ antiporter and was designated as AeNHX1. The AeNHX1–GFP fusion protein was clearly targeted to the vacuolar membrane in a transient transfection assay. Northern analysis indicated that AeNHX1 was expressed in a root-specific manner. Expression of AeNHX1 in yeast Na⁺/H⁺ antiporter mutants showed function complementation. Further, overexpression of AeNHX1 promoted salt tolerance of Arabidopsis plants, and improved osmotic adjustment and photosynthesis which might be responsible for normal development of transgenic plants under salt stress. Similarly, AeNHX1 also functioned in transgenic Festuca plants. The results suggest that this gene might function in the roots of Agropyron plants, and its expression is involved in the improvement of salt tolerance.     

拟南芥ANAC055基因突变体对高盐和渗透胁迫的响应

高盐和渗透等非生物胁迫是影响农作物产量和品质的重要因素,非生物胁迫发生时,植物通过体内各类转录因子启动胁迫应答反应,进而降低非生物胁迫对植物的损伤。本研究筛选出植物特异性转录因子ANAC055编码基因的纯合T-DNA插入突变体SALK_152738,测序分析发现T-DNA插在ANAC055基因的3'UTR区域。实时荧光定量PCR结果表明叶中ANAC055基因表达量最高;与野生型相比,突变体叶、茎和花中ANAC055基因表达量分别下降了40%、50%和70%。高盐胁迫后,野生型和突变体叶中ANAC055基因表达量分别比对照上升了320%和55.4%;而渗透胁迫时,该基因叶中的表达量分别比对照下降了47.7%和56.3%;电子表达谱分析发现该基因根中的表达可受高盐和渗透等多种非生物胁迫的诱导表达。高盐和渗透胁迫时野生型和突变体幼根的生长均受到明显抑制,但高盐胁迫对突变体根生长的抑制作用比对野生型根生长的抑制作用更大。上述分析表明拟南芥ANAC055基因可受高盐和渗透等非生物胁迫的诱导表达,并且其在拟南芥幼根的生长发育过程中具有一定的作用,本研究有助于进一步明确其在非生物胁迫过程中的作用。