Hydroxyproline-rich cell wall protein (extensin): Biosynthesis and accumulation in growing pea epicotyls

Plant cell walls contain a glycoprotein component rich in the otherwise rare amino acid hydroxyproline. We examined the synthesis and accumulation of wall hydroxyproline during different states of elongation growth in pea epicotyls. Light-grown peas contained more wall hydroxyproline than their taller, dark-grown counterparts. When elongation was studied by marking growing stems in situ, there was a marked accumulation of wall hydroxyproline coincident with the cessation of elongation. Dividing and elongating regions of the epicotyl showed less wall hydroxyproline than did regions where elongation was no longer occurring.Hydroxyproline biosynthesis was examined by incubation of excised sections of tissues in various growth states in ¹⁴C-proline. The extent of conversion of these residues to ¹⁴C-hydroxyproline served as a measure of the rate of hydroxyproline synthesis. This rate was highest in tissues which had ceased elongation. The low rate of hydroxyproline synthesis in dividing and elongating cells was probably not due to the inability to hydroxylate peptidyl proline or to secrete proteins.These data show a positive correlation between the synthesis and accumulation of cell wall hydroxyproline and the cessation of cell elongation in pea epicotyls.     

Changing arabinosylation patterns of wall-bound hydroxyproline in bean cell cultures

The arabinosylation patterns of wall-bound hydroxyproline in Phaseolus vulgaris L. cell suspension cultures were determined by separating free hydroxyproline and hydroxyproline-arabinose oligomers over a Bio-Gel P-2 column. Total hydroxyproline accounted for about 3.3% of wall dry weight during all growth phases of batch-cultured bean cells. The chemical arabinosylation patterns of wall-bound hydroxyproline varied during the lag phase and early log phase of the culture. First, an increase in nonglycosylated hydroxyproline occurred accompanied by a corresponding decrease in hydroxyproline tetra-arabinoside. During the early log phase the reverse happened. In later stages of growth the chemical arabinosylation patterns remained constant. The radiochemical arabinosylation patterns were also determined, after pulselabeling the cultures with [¹⁴C]proline at various times during growth, to be able to distinguish recently incorporated hydroxyproline. The time course of the arabinosylation pattern of this fraction indicated that the initial changes in the chemical pattern were due to the temporary incorporation of less extensively glycosylated hydroxyproline-containing protein into the cell wall.Abbreviations Hyp hydroxyproline - HA_n hydroxyproline arabinoside - with n arabinosyl residues - TFA trifluoroacetic acid     

Influence of proline and hydroxyproline on salt-stressed axillary bud cultures of two varieties of potato (Solanum tuberosum)

Summary The effects of exogenously supplied proline and hydroxyproline on the potato (Solanum tuberosum) varieties L.T.8 and Desiree were studied using axillary bud cultures both in the presence and absence of 0.6% salt stress. In both varieties, the effects of exogenously supplied proline and hydroxyproline at 1.0 mM and 2.5 mM were less severe than 0.6% salt alone. At the same time, the accumulation of proline, protein, carbohydrates, sodium, and potassium were similar. However, when both the salt and proline/hydroxyproline were supplied, proline and hydroxyproline provided some measure of protection against salt stress. It is believed that increased proline levels in L.T.8 and increased carbohydrates in Desiree due to the presence of exogenously supplied proline/hydroxyproline were responsible for the additional protection against salt stress in the axillary bud cultures of these varieties.     

Moderately increased constitutive proline does not alter osmotic stress tolerance

Proline-overproducing carrot cell lines were isolated by selection in medium containing hydroxyproline, a toxic analogue of proline. During growth of the cells in culture, length of lag phase, doubling time, and maximum fresh weight were the same for the hydroxyproline-resistant cell line (HP) and the wild-type cell line (JW). Proline content and resistance to hydroxyproline in the HP and JW lines were not strictly correlated indicating that another reason besides the constitutive level of proline is involved in hydroxyproline resistance. Tolerance to polyethylene glycol-induced desiccation stress was not different between the two lines except perhaps at the early stages of culture growth when the proline levels of the two cell lines were nearly the same. The complexity of the relationship between proline accumulation and osmotolerance is discussed and strategies to achieve constitutive high levels of proline accumulation in plants are proposed.     

Effect of IAA on the Transport of Hydroxyproline-protein(s) in Zea Coleoptiles

Indole-3-acetic acid (IAA) induced the accumulation of ¹⁴C-hydroxyprolinein the endoplasmic reticulum (ER)- and Golgi-rich fractions;especially in the ER fraction. It inhibited the incorporationof hydroxyproline in the cell wall fraction. In addition, IAAhad no effect on the ratio of hydroxyproline to proline in eachfraction. The decrease in hydroxyproline content per sectiontreated with IAA was very little. Based on these results wepropose that IAA inhibits the transport of hydroxyproline-proteinin the cell wall, but the resulting decrease in hydroxyproline-proteinrigidly bound to the cell wall may not be the main factor thatinduces cell elongation. (Received July 13, 1981; Accepted February 15, 1982)     

Proline-14C metabolism in barley seedlings during vernalization

The metabolism of proline-¹⁴C was examined in vernalized (LT)and unvernalized (HT) barley shoots. The content of prolinein cytoplasmic and cell wall protein fractions was a littlehigher in HT, against the accumulation of free proline in LT.Proline-¹⁴C was more heavily incorporated into HT and the cytoplasmicprotein had a higher radioactivity than the cell wall protein.In LT, the activity in the cytoplasmic protein was lower thanthat in the cell wall. The time course of incorporated proline-¹⁴Cshowed no distinct changes in HT. but decreased remarkably inthe LT cell wall. The distribution of proline-¹⁴C in hydroxyproline and otheramino acids in the two proteins was examined. In the cytoplasmicproteins, the conversion pattern of proline-¹⁴C was very similarin both treatments. The highest hydroxyproline activity wasfound at the beginning of incubation and was maintained at acomparatively high level in the HT cell wall. The LT cell wallshowed a gradual increase in radioactivity due to hydroxyprolineand reached maximum conversion at the last incubation period.Distribution of radioactivity due to incorporated proline-¹⁴Cwas examined by separating barley shoot tissues into three sections. (Received December 5, 1972; )     

Studies on the biochemistry and fine structure of silica shell formation in diatoms

In diatoms, the siliceous cell walls are enveloped by an organic component which includes 4-hydroxyproline and 3,4-dihydroxy-L-proline. The formation of these two amino acids were studied in Nitzschia angularis in Si-starvation synchrony. Both appear to arise from peptidyl proline. Its conversion to peptidyl hydroxyproline was shown in cell-free extracts and in kinetic studies using [¹⁴C]proline. Two lines of evidence indicate that dihydroxyproline does not arise from the further hydroxylation of peptidyl hydroxyproline: First, there was a lag of several minutes between the incorporation of [¹⁴C]proline into protein and the appearance therein of [¹⁴C]hydroxyproline but no such lag for the appearance of dihydroxyproline. Second, ,-dipyridyl blocked the formation of hydroxyproline, but not of dihydroxypyroline, from peptidyl proline. Cell walls made in the presence of dipyridyl differed little in overall chemical composition from walls made in its absence and were morphologically identical. [¹⁴C]dehydroproline was rapidly metabolized in the cells, with [¹⁴C]dihydroxyproline a prominent product. Studies of the conversion of [¹⁴C]proline to [¹⁴C]hydroxyproline at various stages of wall formation showed an increased synthesis of [¹⁴C]dihydroxyproline at the end of cell separation.     

HRGP在小麦抗寒锻炼过程中的变化及其与抗寒性的关系

强抗寒小麦品种(R-025、中品94-19、品83-1、品83-2、品83-3、米罗诺夫808)经抗寒锻炼后,其幼苗体内的游离脯氨酸、细胞壁结合的羟脯氨酸和糖蛋白含量发生了明显的变化.游离脯氨酸含量比未经抗寒锻炼处理时增加5～32倍,细胞壁结合的羟脯氨酸含量比对照增加1.77～2.17倍,糖蛋白含量比对照增加4.68～9.72倍,而不抗寒小麦品种(中国春、冬103)增加量较小.脯氨酸积累进程各个品种间差异比较大,品83-1、品83-2积累较快,抗寒锻炼第21d时达到最高峰,而R-025在第56d达到最高峰.脯氨酸含量与小麦品种抗寒性相关不显著(相关系数为0.3462),而羟脯氨酸含量、糖蛋白含量与小麦品种抗寒性相关显著,相关系数分别为0.6491和0.7039.从小麦细胞壁纯化得到了2种伸展蛋白Extensm1和Ex-tensin2,其含量都和小麦品种抗寒性呈正相关.Extensin1是分子量为28kD、羟脯氨酸为主要成份(32mo1%)的富含羟脯氨酸糖蛋白.     

Comparison of proline accumulation in two mediterranean shrubs subjected to natural and experimental water deficit

The effect of water stress on proline accumulation was tested in two contrasted species of Mediterranean scrub: Halimium halimifolium (L.) Willk and Pistacia lentiscus L. Leaf water potential, stomatal resistance and proline content have been measured both in experimental and in natural water stress conditions. Both species accumulated proline in their leaves when leaf water potential dropped below a threshold value of –3.0 MPa, under natural as well as under experimental conditions. In the field, however, a time-lag between decrease of leaf water potential and proline accumulation could be observed. In Halimium halimifolium, proline accumulation appeared to be associated with severe stress conditions as most plants with high proline contents suffered irreversible wilting, especially in the greenhouse. P. lentiscus showed a different pattern, accumulating proline at two different times of the year, as a response to cold or to drought. The results of our study indicated that the role of proline in this species, rather than an osmotic agent, seems to be more related to a protective action in cases of severe stress conditions.     

The effect of NaCl on proline accumulation in rice leaves

The regulation of proline accumulation in detached leaves of rice(Oryza sativa cv. Taichung Native 1) was investigated.Increasing concentrations of NaCl from 50 to 200 mM progressivelyincreased proline content in detached rice leaves. NaCl induced prolineaccumulation was mainly due to the effect of both Na⁺ andCl^– ions. Proline accumulation caused by NaCl was related toprotein proteolysis, an increase in ornithine--aminotransferaseactivity,a decrease in proline dehydrogenase activity, a decrease in prolineutilisation,and an increase in the content of the precursors of proline biosynthesis,ornithine and arginine. Results also show that proline accumulation caused byNaCl was associated with ammonium ion accumulation.     

Evaluation of Abiotic Stress Resistance in Mutated Populations of Cauliflower (Brassica oleracea var. botrytis)

An efficient in vitro screening method has been developed for cauliflower to create NEU and NMU induced mutant lines selected on hydroxyproline containing medium. Mutant lines and control plants were sub-cultured many times on maintenance medium and stored at 5°C for 2 years and then tested for salt and hydroxyproline resistance as in vitro and in vivo plants. In vitro shoot tips were also sub-cultured to media containing hydroxyproline and NaCl for 28 days and then assessed for their leaf proline content. Non-acclimated and acclimated in vivo plants were also assessed for resistance to freezing. Populations of control and selected lines were created by mass pollination and subsequently tested for their NaCl and frost resistance. Control plants had little or no NaCl or hydroxyproline resistance whilst selected plants showed varying degrees of resistance. In vitro and in vivo responses of selected lines were correlated. Leaf proline content was increased markedly in the mutant lines and the greatest proline contents occurred following NaCl stress with the most respondent line having 100-fold levels compared to the controls. Both non-acclimated and acclimated selected lines showed improved frost resistance over controls. Improvements in frost resistance were heritable but improvements in NaCl resistance were not. The results clearly demonstrated that NaCl, frost and hydroxyproline resistance were stable traits over repeated in vitro sub-cultures and prolonged low temperature storage. A complete range of mutants with single, double or triple resistance traits were produced. The level of resistance however was not necessarily correlated with the level of proline and some lines showed resistance without elevated proline. It is concluded that elevated proline is not essential for improved resistance to abiotic stress in cauliflower, but where it does occur it does improve resistance.     

Hydroxyproline and Peroxidases in Cell Walls of Pisum sativum: Regulation by Ethylene

Purified cell-wall preparations from the epicotyl of etiolatedPisum sativum contain covalently bound peroxidases and hydroxyproline-richproteins. Towards the end of cell elongation there is a largerise in these wall components and thereafter a continuing slowrise which is associated with increasing age of tissue. Ethyleneat concentrations of 0.1 ppm or more increases both peroxidaseactivity and hydroxyproline levels in the walls, the greatestresponse occurring in immature tissue including the apical hook.Growth of these tissues is highly sensitive to ethylene whichcauses an inhibition of elongation in extending cells and anenhanced lateral cell expansion. We suggest that the effectsof ethylene on wall-bound peroxidase and hydroxyproline areimplicated in the ethylene regulation of cell growth. The covalently bound wall peroxidase was found to be extremelystable and to contain unique isoenzymes which do not occur ineither the cytoplasm or in the peroxidase which is ionicallybound to walls. Ethylene increases peroxidase activity in boththe cytoplasmic and the ionically bound wall fractions, butthere is little or no increase in their hydroxyproline content.The possible relationships between covalently bound wall peroxidaseand hydroxyproline are discussed and we speculate that thisperoxidase may be involved in the hydroxylation of proline inthe walls.     

Studies on Isolation and Characteristics of a Sugarcane Cell Line Resistant to Hydroxyproline (HYP)

Cultured sugarcane(Saccharum sinensis Roxb.) cell line R8968 was selected for resistance to growth inhibition by hydroxyproline(HYP), a proline analog. Comparison of the free amino acid pool level in the line with that of the HYP-sensitive donor showed substantial accumulation of proline(3.8 × ). The responses of the cell line R8968 to NaC1, freezing temperature and PEG were also compared with those of the donor and the former exhibited remarkably increased tolerance to the tested conditions. The results showed that proline accumulation may favour the increased tolerance.     

Distribution and metabolism of protein-bound hydroxyproline in an elongating tissue, the Avena coleoptile

A study has been made of the distribution and metabolism of protein-bound hydroxyproline in an elongating tissue, the excised Avena coleoptile. The hydroxyproline-containing proteins of this tissue have been separated into 3 fractions on the basis of their solubilities. The cytoplasmic, trichloroacetic acid-insoluble proteins (S-fraction) contain the bulk of the proline of the cells but only 20% of the hydroxyproline. The cytoplasm also contains a previously unrecognized trichloroacetic acid-soluble, non-dialyzable fraction (DS-fraction) which is low in proline but contains 20% of the hydroxyproline. The remaining 60% of the hydroxyproline is in the wall-bound, cold alkali-soluble fraction (extensin).

Incorporation of free proline into the proline and hydroxyproline of all fractions is linear with time for at least 12 hours. The specific activity of the proline at any time is the same in all 3 fractions while the specific activity of the hydroxyproline is 4-times greater in the S-fraction than in the W-fraction. During a pulse-chase experiment the specific activity of the proline decreases 25 to 40% in all fractions during the chase. The labeling of hydroxyproline in the wall increases during the chase while that of the DS-fraction remains constant. In the S-fraction, the labeling in hydroxyproline rapidly drops 30 to 35% during the chase but then remains constant. It is concluded that the majority of the hydroxyproline-proteins in the cytoplasm are not transported to the wall. It is suggested that a sizeable portion of the cytoplasmic hydroxyproline may be located in enzymatic proteins.

     

Effects of hydroxyproline on the growth and cell-wall protein metabolism of excised root segments of Pisum sativum

Summary Hydroxyproline, in the presence of sucrose, enhanced the extension growth of excised 2–4 mm pea root segments in aseptic media. About 90% of protein-bound hydroxyproline in the pea root segments was confined to the cell-wall fraction where it occurred as trans-4-hydroxy-l-proline. The amounts of wall-bound hydroxyproline increased dramatically towards the cessation of extension growth, but when the segments were cultured in trans-hydroxyproline, this increase was considerably less.Externally supplied cis and trans-hydroxyproline inhibited the formation of protein-bound [¹⁴C]hydroxyproline from [¹⁴C]proline without affecting the total amount of [¹⁴C]proline incorporated into proteins. Studies with -dipyridyl showed that, although some of the externally supplied trans-[¹⁴C]hydroxyproline was incorporated directly into cell-wall proteins, most of it was first converted into proline which was then incorporated into proteins and subsequently reconverted, in part, into hydroxyproline. The effect of externally supplied hydroxyproline is discussed in relation to protein-bound proline hydroxylation.     

Transport of Proline and Hydroxyproline by the Neutral Amino-acid Exchanger ASCT1

ASCT1 is a member of the glutamate transporter superfamily cloned from human brain and characterized as a Na⁺-dependent neutral amino-acid exchanger, which displays substrate-induced chloride-channel activity and mediates concentrative transport of alanine. Initial studies in ASCT1-expressing Xenopus laevis oocytes showed that proline did not elicit measurable currents, in contrast to what occurred with alanine, serine or cysteine, suggesting that proline was not an ASCT1 substrate, although it induced the release of alanine from preloaded oocytes. Here, we have studied the uptake of proline and hydroxyproline by ASCT1-expressing oocytes in order to investigate the ability of ASCT1 to translocate these imino acids. The results demonstrate ASCT1-mediated proline transport that is Na⁺-dependent, saturable, inhibited by the reported ASCT1 substrates as well as by hydroxyproline and can drive the imino acid against its concentration gradient. The apparent kinetic constants for the transport of alanine and the imino acids, obtained with oocytes from the same batch, showed maximal transport rate for proline and hydroxyproline to be half of that for alanine. However, K _0.5 for proline was 704 ± 86 µM, about three times higher than alanine K _0.5 (203.3 ± 36.4 µM), whereas hydroxyproline K _0.5 was 33.2 ± 4.3 µM, indicating that the hydroxylation on carbon 4 of proline strongly increases the affinity of ASCT1 for this proline derivative. In summary, the present work demonstrates for the first time the ability of ASCT1 to transport proline and hydroxyproline.     

Salt stress enhances proline utilization in the apical region of barley roots

Accumulation of an osmoprotectant, proline, is enhanced in response to salinity in plants. Here, by immunohistochemical analysis, we demonstrated that proline transporter (HvProT) was highly expressed in the apical region of barley roots under salt stress. Free proline was accumulated more in the basal region than in the apical region of barley roots under salt stress, although expression level of HvProT was higher in the apical region. On the other hand, salt stress increased proline and hydroxyproline contents in the cell wall fraction of the root apical region, suggesting increment of proline utilization. Expression of the genes encoding cell wall proteins (proline rich protein and extensin) and cellulose synthase was induced in barley roots by salt stress. These findings indicated that free proline transported by HvProT presumably behaved as a component of cell wall synthesis in the apical region of barley roots under salt stress.     

Inhibition of formation of protein-bound hydroxyproline by free hydroxyproline in Avena coleoptiles

Free hydroxyproline inhibits the formation of protein-bound hydroxyproline from proline to a considerably greater extent than it does the incorporation of proline into protein of auxin-treated Avena coleoptiles. This inhibition is greater in the wall than in the cytoplasmic fraction. In the absence of auxin, free hydroxyproline exerts little or no inhibition of hydroxyproline formation. Furthermore free hydroxyproline has no effect on respiration, RNA synthesis or the incorporation of leucine into protein. Hydroxyproline is not a general inhibitor of metabolism or protein synthesis in Avena coleoptiles.

These results suggest that free hydroxyproline may inhibit auxin-induced cell elongation by blocking the formation or utilization of a particular hydroxyproline-rich protein which must be incorporated into the cell wall during auxin-induced wall extension.

     

Al-induced cell wall hydroxyproline-rich glycoprotein accumulation is involved in alleviating Al toxicity in rice

Cell wall components such as pectin and hemicelluloses have been proposed to be involved in aluminum resistance mechanisms in plants. However, whether hydroxyproline-rich glycoproteins (HRGPs), one of the most abundant proteins of the cell walls, are involved in Al resistance mechanisms remains elusive. In this study, two rice cultivars Xiushui 03 (Al resistant) and Xiushui 128 (Al sensitive) significantly differing in Al resistance were identified. In the absence of Al, no significant difference was observed in contents of glycoproteins and hydroxyproline in cell wall fractions of these two cultivars. At the early stage of Al toxicity, glycoproteins and hydroxyproline were significantly induced in these two cultivars, but levels of their accumulation in cell walls were much higher in cv. Xiushui 03 than in cv. Xiushui 128. At the late stage of Al toxicity, their accumulation in cell walls dramatically decreased in cv. Xiushui 128 and, however, still kept a high level in cv. Xiushui 03. The finding that Al-induced changes of glycoproteins and hydroxyproline were completely consistent indicates that Al-induced glycoproteins are HRGPs. Further observation utilizing transmission electron microscope showed that HRGPs were greatly accumulated in cell walls leading to thickening of cell walls in cv. Xiushui 03, however, HRGPs and cell walls greatly decreased in cv. Xiushui 128. These data suggest that Al-induced HRGP accumulation in cell walls is involved in alleviating Al toxicity in rice.     

Synthesis and Transport of Hydroxyproline-rich Components in Suspension Cultures of Sycamore-Maple Cells

Plant cell walls contain a glycoprotein rich in hydroxyproline. To determine how Acer pseudoplatanus L. cells transport this glycoprotein to the wall, the pulse-chase technique was used to follow changes in specific radio-activity of hydroxyproline and proline in isolated, mitochondrial, Golgi, microsomal, soluble protein, and wall fractions. The turnover rates or changes in specific radioactivity of cytoplasmic hydroxyproline in these cell fractions indicated that the bulk of this hydroxyproline was transferred not by the Golgi apparatus but by a smooth membranous component.